Dye-coupling among neurons of the rat locus coeruleus during postnatal development

NeuroscienceVol. 56, No. 1, PP. 129137, 1993 Printed in Great Britain

DYE-COUPLING COERULEUS

AMONG DURING

0306-4522/93$6.00+ 0.00 Pergamon Press Ltd 0 1993IBRO

NEURONS OF THE RAT LOCUS POSTNATAL DEVELOPMENT

M. J. CHRISTIE* and H. F. JELINEK Department of Pharmacology, The University of Sydney, N.S.W. 2006, Australia Abstract-Simultaneous recordings from pairs of locus coeruleus neurons in neonatal rat brain slices previously demonstrated synchronous, subthreshold oscillations of membrane potential (rats < 24 days old) and electrotonic-coupling between 40% of pairs of neurons from rats less than 10 days old.3 In the present study, slices from 1-21 day-old rats were stained with avidin-HRP-diaminobenxidine only if a single neuron per slice was impaled for longer than 10 min using an electrode containing biocytin. In slices from rats less than one week old, multiple stained neurons (3.8 f 0.6 neurons/slice) were observed in 10 of 11 slices. Apparent contacts between stained neurons were observed at varying distances along dendrites. In rats older than one week significantly fewer multiple stained neurons were observed (three of 20 slices). The proportion of neurons displaying spontaneous subthreshold oscillations of membrane potential decreased with age, and the frequencies of subthreshold oscillations of membrane potential and entrained action potentials increased with age. The presence of multiple stained neurons was not correlated with the occurrence of subthreshold oscillations, cell input resistance, or the number of coupled neurons predicted from the shape of electrotonic potentials. In recordings from neurons displaying subthreshold oscillations, input resistance was lower and the number of coupled neurons predicted from electrotonic potentials was greater than in those without oscillations. These results suggest that low resistance pathways are common between locus coeruleus neurons in brain slices from rats younger than about one week old, consistent with previous electrotonic-coupling studies. Coupling between neurons may be partly responsible for synchronous oscillations, and could contribute to the widespread trophic role of noradrenergic neurons during early development.

The locus coeruleus (LC) is the largest group of noradrenergic neurons in the mammalian central nervous system. 24 Considerable evidence suggests a regulatory role for the LC during development.23*25In the rat, the LC is the first monoaminergic cell group to differentiate (post-gestational days 1@13).‘6*32Innervation of cerebral and cerebellar cortices by LC afferents precedes differentiation of these regions, being detectable by gestational days 16-17,23,25and LC neurons can be antidromically activated from cortex at least as early as gestational day 18.” Furthermore, lesion studies suggest a trophic role for noradrenaline in cortical development,’ and in maintenance of synaptic plasticity in the developing visual cortex,i4 rat barrel cortex” and olfactory bulb.33 LC neurons undergo functional changes during development which might be crucial to their trophic role.23,2s Recordings from neonatal LC neurons in brain slices display calcium-dependent, subthreshold oscillations of membrane potential,36 which are synchronous throughout the entire nucleus.) The frequency of oscillations increases from -0.3 to 3 Hz and amplitude decrease until -four weeks of age when they are infrequently observed.3s36 This property could contribute to the synchronous activity, fluctuations in excitability and sensory responsiveness reported in neonatal LC neurons in Y~uo.‘~Syn*To whom correspondence should be addressed. Abbreviations:

oxidase.

LC, locus coeruleus; HRP, horseradish per-

chronous oscillations could also contribute to the trophic role of the LC by entraining responsiveness to synaptic inputs and the release of noradrenaline throughout the central nervous system.3.23 In brain slices and explant cultures, LC neurons are also stimulated by a,-adrenoceptor agonists, in contrast to adult neurons which show little or no response.8.36 The functional roles of changing adrenergic responsiveness in LC neurons and their targets are not known.*) Direct coupling between LC neurons via low-resistance pathways such as gap-junctions could be one of the mechanisms responsible for the high synchrony of membrane potential oscillations in neonatal LC3 Direct electrotonic-coupling was previously reported in 41% of pairs of LC neurons (12 of 29 pairs) in slices from animals less than IO days old, but not in several slices from older rats (only four pairs were tested).3 The present study was designed to estimate the extent of coupling between LC neurons during postnatal development utilising the intracellular diffusion of biocytin, a low molecular weight, water soluble stain.” Biocytin (molecular weight = 373) can presumably pass through gap-junctions between neurons, and has been used by others to demonstrate junctions between neonatal hippocampal neurons.5 This method is technically more straightforward than dual impalements and the extent of coupling between neurons should not be influenced by cell packing density or the separation between two electrodes. In the present study, membrane properties were also 129

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M. J.

CHRISTIE

measured to investigate the relationship between the presence of coupled networks and changes in electrophysiological properties during development of the LC neurons. EXPERIMENTAL PROCEDURES

Intracellular recordings were made from LC neurons essentially as previously described.’ Briefly, Wistar rats of either sex were used (University of Sydney Animal Services), in the postnatal age range 1-21 days. They were anaesthetised with halothane and killed by decapitation. Horizontal sections containing pons (400 pm thick) were prepared using a vibratome. Slices containing the LC (2-3 slices/ animal) were bisected along the mid-line and maintained for 057 h completely submerged in a heated (35’Q gassed (95% 0,/5% COI) physiological saline of the following composition (mM): NaCI, 126; KCl, 2.5; CaClr ,2.5; MgCl,, I .2; NaH*PO,, 1.2; NaHCO,, 25; glucose, 1I. For intracellular recording, individual slices were transferred to a recording chamber (300~1) and submerged in heated (35”C), flowing (1.4ml/min) physiological saline. The LC was visible in transilluminated slices as a translucent, crescent-shaped region bordered by the fourth ventricle. Intracellular recordings of membrane potential (Axoclamp HA, Axon Instruments) were made with microelectrodes (3c-80 MR; mean + SD. = 44 _+ 9 mR, n = 31) filled with KC1 (2 M) and biocytin (2%, Sigma, St Louis) which was buffered with 50 mM Tris-HCl (final pH 7.4 at 25”C).‘? Recordings of membrane potential from each neuron were plotted simultaneously on a chart recorder. All data are presented as mean + S.E.M., unless otherwise stated. Considerable care was taken to ensure that only one cell in each slice was impaled with a biocytin filled electrode. Slices were first tested using electrodes containing no biocytin to ensure high viability, as judged by the impalement of several healthy neurons in a single electrode track. Neurons were then impaled with biocytin filled electrodes only in highly viable slices. Electrodes were withdrawn within 30s (usually less than 10s) from cells unless membrane properties were of high quality, i.e. an action potential amplitude greater than 50mV, and frequency less than 10 Hz. Slices were also rejected from further analysis if impalements were maintained for less than 10 min. In these experiments (including preliminary experiments), biocytin filled neurons could be histologically identified in 86% of slices if impalements lasted longer than 10min (37 slices), 38% for 2-10 min (13 slices), and 0% for less than 2 min (six slices). Application of these criteria led to the rejection of -80% of slices from further analyses. In slices meeting the above criteria, impalements were lost or electrodes withdrawn within 65 min (mean & SD. = 37 & 14 min. n = 31). Slices were maintained in the recording chamber for a further 30_50min, then fixed overnight, or longer in phosphate-buffered (0.1 M, pH 7.2) paraformaldehyde (4%) prior to staining for biocytin essentially as described elsewhere. I3 Briefly, after rinsing the fixative, slices were incubated in phosphate buffer containing 0.3% Triton X-100 for at least 4days, and then reacted with avidin-horseradish peroxidase (HRP) complex (Vector Laboratories, Standard Peroxidase kit) for 120 min. Slices were then rinsed for 30min and reacted with diaminobenzidine (Sigma, St Louis) without Nickel intensification. Stained slices were cleared with, and mounted in dimethylsulphoxide. Stained neurons in each slice were counted, and in several cases photomicrographs and camera-lucida drawings were prepared. In some experiments, electrotonic potentials were examined in physiological saline containing CaCl* (0.2 mM) and MgCl, (I 1.2 mM) to eliminate rhythmic variations of membrane potential. j6 Small membrane potential changes of 2-8 mV amplitude and 100-200 ms duration were evoked by

and H. F. JELINEK

current steps of 20-50 pA amplitude from a resting potential of -75 to -85 mV. Averages (50 -100 samples) of 500 points were digit&d at intervals of 2OC-400 /us (“TL- 12.5”. Axon Instruments). Electrotonic potentials w’ere tilted to one or more exponentials (“PCLAMP” software. Axon Instruments). The correlation coefficeints for the fits were tested to determine whether they were significantly different (P c 0.05) before concluding that the electrotonic potentials were better fit by one or two exponentials. In all but one of 16 experiments analysed, two exponentials better titted the data, with correlation coefficients greater than 0.995. The method of Publicover” was then used to predict the number of neurons present in a coupled network from the amplitude and time constant parameters of these exponential components. In this model, cell membranes are represented by a parallel resistance (R,) and capacitance (C,), connected by coupling resistances (R,). The total electrical load of the coupled network is quantified in terms of a number of equivalent cells (N,). This model deviates from previous “lumped soma, short cable” models?” by describing these coupled cells in terms of a membrane resistance R’,,,. and capacitance C’,, which represents an equivalent coupled cell, although the form of the responses (i.e. two time constants) is the same as in previous models, The conmutational formula is:

Tm R,, A,, r,

N, = I1;,:/,

‘

where N, is the number of coupled cells, A,. A,, T, and 7; are the primary and coupled amplitude and time constant terms, respectively. In small networks, the membrane resistance, R, and the coupled resistance, R’, can be assumed to he approximately equal.

RESULTS

Appearance

of’ neurons stained with biocytin

Examples of the appearance of slices containing multiple biocytin stained neurons are presented in Figs l-3. In these, and most other cases, large multipolar neurons were stained, although fusiform neurons were sometimes observed. Soma diameters appeared to range from 20 to 4Opm, but were difficult to estimate because dimethylsulphoxide produces considerable swelling of slices. Although the intensity of the HRP reaction product appeared to vary between neurons, this did not appear to be related to depth within stained slices. Neurons often had dendrites which branched once or twice and appeared to extend beyond the margins of the LC. However, the borders of the nucleus were not readily discernible, even in counterstained material. Dendrites occasionally displayed a “swollen” appearance at, or near the surface of the slice. Dendritic spines were usually (e.g. Fig. 2), but not always (e.g. Fig. 3) visible. In several cases, stained axons extending several mm beyond the LC were observed. The intensity of staining appeared to be uniform throughout each neuron’s arborisation. Fig. la shows an example of coupled neurons taken from a five-day-old rat, and Fig. lb shows two superimposed neurons from a three-day-old rat (a third neuron was outside of the plane of focus). In most cases, multiple stained neurons could not be photographed because they were not contained within the same focal plane. Fig. 2 shows a camera

Fig. 1, Photomicrographs of multiple a~din-H~~iamino~nzidine stained neurons after impalement of a single neuron in each 400~pm-thick horizontal slice using a biocytin filled electrode. In each case, the top of the photograph is rostra1 and the mid-line to the right, with the fourth ventricle near the bottom right corner. (A) slice prepared from a five-day-old rat (see Fig. 2 for camera lucida drawing). (B) slice prepared from a three-day-old rat (a third stained neuron was out of the plane of focus). Scale bar = 50 pm.

M. J. CHRISTIEand H. F. JELINEK

132

Fig. 2. Camera-lucida drawing of avidin-HRP-diaminobenzidine stained neurons after impalement of a single neuron using a biocytin filled electrode in an horizontal slice prepared from a five-day-old rat (same slice as in Fig. la). The top of the drawing is rostra1 and the mid-line is to the right, with the fourth ventricle near the bottom right corner. Scale bar = 50 pm, arrows indicate possible points of contact (i.e. close apposition of membranes).

lucida drawing of the neurons visible in Fig. la. Although the existence of cell-cell junctions cannot be confirmed using light microscopy, several possible points of contact could he seen in this, and most other examples. Points of contact could not clearly he identified in cases where the fields of view of two

somata overlapped (e.g. Fig. lb). Separation between stained somata varied considerably in different slices, from apparently direct somatic contact to over 200pm separation. An example of multiple, widely separated, stained neurons in a slice prepared from a four-day-old rat is shown in Fig. 3. In this case, points of ccllLcel1 contact were less obvious than for

the example shown in Fig. 2. Multiple stained neurons were observed in 13 of the 31 slices examined. Overall, 2.0 &-0.4 neurons/slice were visible (n = 31 slices), usually with possible points of contact (see above). Table 1 shows the relationship between number of neurons visible and age of the animal. Multiple stained neurons were frequently observed in slices from animals less than one week old (10/l 1 slices). In contrast, significantly fewer slices from older animals contained multiple stained neurons (3/20 slices x2, 1 d.f. = 13.9, P < 0.0005, Yates corrected). Electrophysioiogicalproperties biocytin

of neurons stained with

As previously described,3,36 some membrane properties of LC neurons varied with age (Table 1). In the

absence of applied currents, most cells (28/31) fired spontaneous action potentials 6GSOmV in amplitude, with a threshold near - 55 mV. Action potentials usually arose from the peak depolarising phase of subthreshold oscillations in membrane potential (raw data not shown), which were similar to those previously described. 3,36As presented in Table 1, the frequency, but not the occurrence of spontaneous action potentials increased with age. The proportion of neurons displaying subthreshold oscillations decreased with age, and the frequency of oscillations increased with age. Other membrane properties, such as input resistance, were not significantly different. If coupling between LC neurons was responsible for generating and/or synchronising subthreshold oscillations of membrane potential, then slow rhythmic activity might be expected to be more frequent in slices having multiple stained neurons, regardless of age. As shown in Table 2, the proportion of recordings displaying subthreshold oscillations did not significantly differ between slices with multiple stained neurons and slices with single stained neurons. The presence of coupled networks might also explain differences in membrane resistance and the presence of multiple exponential components of electrotonic potentials recorded from neonatal LC neurons3 Electrotonic potentials evoked by small current steps were investigated after elimination of spontaneous activity by superfusion with physiological saline containing a high concentration of Mg”

Dye-coupling among locus coeruleus neurons

Fig. 3. Camera-lucida drawing of avidin-HRP-diaminobenzidine stained neurons after impalement of a single neuron using a biocytin filled electrode in an horizontal slice prepared from a four-day-old rat. The top of the drawing is rostral, with the fourth ventricle shown in the bottom left corner. Scale bar = 50 pm, arrows indicate possible points of contact (i.e. close apposition of membranes).

(11.2 mM) and low Ca*+ (0.2 mM).36 Electrotonic potentials were fitted with one or two exponential components. Correlation coefficients using a least squares minimisation method to fit the sum of two exponentials were greater than 0.995 in fifteen exper-

iments. Fitted parameters and other membrane properties for single and multiple stained neurons are shown in Table 2. The only significant difference was a greater apparent membrane time constant (r,,,) in recordings from multiple stained neurons.

134

M. J. Table

1. Occurrence

I

and H. F. JI-.LINLK

of multiple stained neurons and membrane coeruleus neurons vary with postnatal age

/IMULTISTAINED

Age (weeks)

CHKWIE

IO/l1 3,:20tt

fl SLICE

PSAP

./SAP

IO/l I IX,/20

3.8 * 0.6 2.0 i 0.0

1.2+o.i 1.8 +0.2*

properties

of locus

pso

.fk>

RN,

9II I 9/20t

0.6kO.l 1.7*0.2**

noi 18 x7 + 13

Multiple stained neurons and membrane properties were examined in slices from rats up to three weeks old. pMULTI-STAINED represents the proportion of single impalements with biocytin filled electrodes in which multiple neurons were stained in slices from animals one week old. n/SLICE represents the number of stained neurons observed in each slice (n = 10 for one week). pS0 represents the proportion of neurons displaying spontaneous subthreshold oscillations of membrane potential in each group.& represents the frequency of subthreshold oscillations in membrane potential (Hz; n = 9 for one week). R,, represents input resistance (MR: )? = 6 for one week). ‘P < 0.02, **P < 0.001 (unuaired /-tests. d.f.), tP < 0.05 (X’-test, 1d.f.), ttP < 0.0005 ($tcst. 1 d.f.). ’

and possible sites of cell-cell contact could usually be identified. The present results also suggest that lowresistance pathways occur between neonatal LC neurons predominantly in slices prepared from animals less than one week old. Multiple stained neurons were encountered frequently in slices from animals less than one week old (10/l 1 slices), but infrequently from older animals (3/20 slices). Electrotonic-coupling was previously reported between pairs of simultaneously impaled LC neurons in slices from animals less than 10 days old (12 of 29 pairs), but not in several animals more than 10 days old (none of four pairs).j A likely basis for dye-couping between neonatal LC neurons is direct cell-cell coupling via gap-junctions. Although gap-junctions have not been detected in electron microscopic studies of adult LC,‘” we are not aware of any such studies in neonates. The transient expression of several gap-junction proteins in neonatal central nervous system6 is consistent with the possibility that they are present in LC during a brief postnatal period. Direct cell&cell coupling might also be produced as an artefact of slice preparation. Dye-coupling in guinea pig cortex was proposed to be

Overall, the method of Publicover” predicted, on the basis of time constants and amplitudes of exponential components, that each impaled neuron was coupled to 2.0 f 0.4 (n = 15 impalements) other neurons. There was a non-significant trend (P < 0.1, unpaired t-test) to predict a greater number of coupled neurons in slices containing multiple stained neurons (Table 2). However, when neurons displaying subthreshold oscillations of membrane potential were compared with those that did not, a significantly greater number of coupled neurons was predicted for oscillating neurons (Noscl,,a,ing:2.3 + 0.5; II = IO, N no, oscl,,at,ng: 1.3 f 0.2; n = 5, P < 0.05, unpaired t-test). These neurons also had a lower apparent membrane resistance (N,,,,,,t,,r: 66 + 8 MR, N notoX,,lallng:119 + 20 MQ P < 0.05, unpaired f-test). DISCUSSION

Multiple stained neurons were frequently observed in slices of neonatal LC in which only one neuron had been impaled with a biocytin filled electrode (13/3 1 slices). Stained neurons had a similar morphology to that described in previous Golgi-staining studies,‘4

Table n/slice

%SO

2. Membrane R,,,

properties T,,,

of single and multiple A”,

T<

stained AC

neurons n,

tl=l

50

76,

15

18*2

0.75 F0.05

4.2 If: 0.9

0.25 kO.05

1.6 + 0.3

nkl

69

90 *

15

28 * 4*

0.67&

7.1 & 1.3

0.33

2.3 + 0.6

0.06

kO.06

nubs I 2.9 +0.6

Recordings of membrane potential from slices containing single or multiple stained neurons were analysed for the presence of subthreshold oscillations (SO; n = 18 for slices containing single, and n = 13 for slices with multiple stained neurons). During some recordings (n = 7 for single and n = 8 for multiple stained neurons), electrotonic charging curves were fitted by the sum of two exponential functions using a least squares method. The correlation coefficients of fit were greater than 0.995 in each case. R,, , apparent input resistance (MO); 7’,,,, membrane time constant (ms); T,, coupled time constant (ms); A,, amplitude of T,,,; A,, amplitude of T,. Fractional weightings of A, and A, are presented, where A,,, + A, = 1, to simplify direct comparison of the two groups. The fitted parameters were used to estimate the number of neurons which were electrically coupled to the impaled neuron using the method of Publicover*’ (see Experimental Procedures for details; ncr number of coupled neurons, nabs, number of neurons stained with biocytin). No t-test, 13 d.f.). correlation was observed between n, and nnhr (r = 0.13). *P -c 0.05 (unpaired

Dye-coupling among locus coeruleus neurons an artefact produced by sectioning dendritic arbors and was modified by changing the plane of section.” This seems unlikely because no effect of plane of section was observed in neonatal LC3 although the disk-shaped dendritic fields of LC neurons are oriented largely in the parasaggital plane.” Although considerable care was taken to avoid impalement of more than one neuron in each slice, the possibility that some neurons were spuriously filled with biocytin cannot be excluded. This seems unlikely, however, because stained neurons were not observed following impalements of less than 2 min duration, and slices having multiple impalements lasting longer than 30 s were rejected. Secondly, spurious impalements cannot explain the observation that multiple stained neurons occurred predominantly in slices from animals less than one week old. Finally, close appositions of soma-soma, dendrite-soma or dendrite-dendrite membranes could be identified in most coupled networks. Intracellular diffusion of biocytin might have failed to identify all coupled neurons within networks; i.e. the HRP reaction product might have been too weak to visually identify all coupled neurons, or poor penetration of the avidin-HRP complex might have failed to identify coupled neurons located deep within slices. The latter possibility seems unlikely because arborisations of stained neurons appeared to be uniformly stained throughout slices. In the present study, multiple stained neurons were observed in 69% of slices prepared from animals I 10 days old (raw data not shown), which was similar to the proportion of dual impalements that revealed electronically coupled neurons in a previous study3 (x2 1 d.f. = 3.0 P > 0.05). However, the probability of encountering a coupled neuron during a dual impalement in the previous study3 could have been quite low. If the LC is assumed to contain _ 1500 neurons34 in a volume of 0.15 mm3 (estimated from Ref. 26) then the packing density would be = 10,000 neurons/mm). If a second electrode were to impale a cell within a sphere around the first electrode having a 100 pm radius, the probability of observing electrotonic-coupling between the pair would be 0.024 if the first cell is only coupled to one other cell, and 0.048 if it is coupled to two others. Therefore, biocytin staining would have been expected to reveal a higher frequency of coupled neurons than dual impalement studies,3 had diffusion of biocytin identified all coupled neurons. Membrane properties of LC neurons displayed several novel features during postnatal development. The proportion of neurons displaying subthreshold oscillations decreased with age, and the frequencies of subthreshold oscillations of membrane potential and entrained action potentials increased with age (Table 1). Subthreshold oscillations were previously reported to increase in frequency and decrease in amplitude during l-28 days of age.3,36 Input resistance did not significantly differ between animals

135

one week old (Table 1). Input resistance was similar to values reported previously,3 but lower than that found for adult cells in earlier studies (201 + 21, n = 19,37213 + 33, n = 7’). Overall, each neonatal LC neuron was coupled to one other biocytin stained neuron (1 .O + 0.4, n = 3 1). Where coupled neurons were identified in animals up to one week old, the extent of the stained network was 3.8 f 0.6 neurons (n = 10). Analysis of the shape of electrotonic potentials evoked by rectangular current pulses2’ suggested a similar number of coupled neurons (Table 2). However, the extent of the coupled network predicted from electrotonic potentials did not significantly differ between slices containing single or multiple stained neurons. This is not surprising because the analysis is based on several assumptions, which could have distorted the analysis if invalid.27 Firstly, estimates based on the shapes electrotonic potentials cannot resolve the electrical load of coupled neurons from that contributed by dendrites. It is possible that dendrites proliferate as electrotonic coupling decreases with age, leading to a similar electrical load in both cases. Secondly, the model assumes that coupled elements have similar electrical properties to the impaled neuron. Direct cellcell coupling might be responsible for maintaining the synchrony of membrane potential oscillations previously reported to occur in neonatal LC up to at least the 24th day,3 and occasionally in adults.‘* Although multiple neuronal staining was not invariably related to the occurrence of oscillations, neurons displaying oscillations had a lower apparent membrane resistance than those without, and the shape of electrotonic potentials predicted a larger apparent number of coupled neurons. Both of these membrane properties could be a consequence of a larger dendritic load and/or a large coupled network. In contrast, electrotonic potentials recorded from adult LC neurons were fitted well by a single exponential component,3 and displayed a higher input resistance (see above). Failure to observe dye-, or electrotonic-coupling in animals older than about one week suggests that mechanisms other than cell-cell coupling might contribute to entrainment of oscillations. Similar subactivity (not threshold rhythmic necessarily synchronous) was reported in up to 30% of LC neurons from 33-37-day-old rats,35 and occasionally in slices from older rats.12.36*37 Other possible explanations, such as synchronous activity of synaptic inputs were ruled out in neonatal LC.3,36 Ephaptic interactions were proposed to possibly contribute to synchrony among LC neurons in explant cultures.’ Synchronous inhibitory synaptic potentials, presumably arising from dendro-dendritic release of noradrenaline, have been reported to induce synchronous subthreshold oscillations in adult LC, in the presence of cocaine. I2 The u,-adrenoceptor antagonist, idazoxan, abolished these oscillations. However, such dendro-dendritic release of noradrenaline can-

136

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CHRISTIE

not fully explain synchrony in neonates, because idazoxan (and prazosin) failed to affect oscillations in slices from neonatal rats.36 Cell-cell coupling might contribute to the synchronous activity of LC neurons early in development, while other mechanisms, such as synchronous inhibitory postsynaptic potentials,” or synchronous discharge of afferents, become more important as synaptogenesis proceeds” during the first weeks of postnatal development. It is not known whether synchronous oscillations also occur in the neonatal IX in uivo, but several observations support the possibility. Firstly, extracellular recordings from LC neurons in perinatal rats in uivo displayed sporadic bursts of activity’5,2Ywhich were synchronous throughout the entire nucleus. This activity could have been entrained by synchronous subthreshold oscillations of membrane potential. The amplitude of the field response evoked by stimulation of the dorsal noradrenergic bundle also varied with time. The amplitude of this field potential is dependent on the number of LC neurons activated, and could vary substantially depending on the timing of the stimulus with the phase of slow depolarisations. Finally, foetal LC neurons were reported to respond vigorously to sensory stimulation,r5 although synaptic contacts are sparse in LC until the second postnatal week, and synaptogenesis continues into adulthood.” Coupling between cells would be expected to increase the number of LC neurons responding to sensory input. Both subthreshold oscillations of membrane potential and low resistance junctions have been reported to occur primarily in the developing central nervous system. Subthreshold oscillations might function to coordinate action potential activity of populations of neurons. In inferior olivary neurons of the guineasynchronous calcium-dependent oscillations pig, 1*‘9-2L entrain action potentials at the peaks of slow depolar-

and H. F.

JEL.INIX

isations, and may play a role in motor coordination. Dye-coupling was reported to be more frequent m neonatal rat cerebral cortex than in adults.” Spontaneous oscillations of membrane potential with properties similar to those reported here,3’ as well as dye-coupling ‘” have been reported in sympathetic preganglionic neurons in the intermediolateral nucleus of the neonatal rat. In the CA3 region of the neonatal hippocampus, low-resistance junctions” might function to synchronise periodic inward currents which can be observed under some conditions.’ CONCLUSIONS

The main finding of the present study was that a large proportion of LC neurons were dye-coupled in brain slices which were prepared from rats during the first postnatal week. Coupled neurons were observed infrequently in slices prepared from older animals. The decline in dye-coupling was paralleled by an age-related decline in the occurrence, and (when present) an increase in the frequency of subthreshold oscillations in membrane potential. However, the presence of dye-coupled networks was not directly related to the occurrence of subthreshold oscillations. Synchronous, subthreshold oscillations may be entrained by low resistance junctions in young animals, whereas other mechanisms. such as synchrony of slow inhibitory synaptic potentials (dcnro-dendritic),j2 could become more important in older animals. The functional significance of synchronous oscillations of membrane potential in neonatal LC neurons has yet to be established, but could be involved in the trophic intluence of the I-C during development. Acknow~edgemenfs--Supported by the National Health and Medical Research Council of Australia (no. 910831). and the Clive and Vera Ramaciotti Foundation.

REFERENCES

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Dye-coupling among locus coeruleus neurons 12. Harris C. G., Hauksen Z. E. and Williams J. T. (1992) Cocaine noradrenergic neurons in vitro. Neuroscience So, 2.53-257.

induced synchronous

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2 April 1993)