501

J. Physiol. (1975), 246, pp. 501-538 With 22 text-figure8 Printed in Great Britain

THE FUNCTIONAL STATUS AND COLUMNAR ORGANIZATION OF SINGLE CELLS RESPONDING TO CUTANEOUS STIMULATION IN NEONATAL RAT SOMATOSENSORY CORTEX S1

BY MICHAEL ARMSTRONG-JAMES From the Physiology Department, London Hospital Medical College, Turner Street, London El 2AD

(Received 27 February 1974) SUMMIARY

1. An investigation was carried out on single cells in 7 day old rat primary somatosensory cortex, which responded to cutaneous stimulation using mechanical pulses. 3 % of cells encountered showed stable spontaneous activity, whereas 88 % were silent in the absence of intentional stimulation. The remainder showed unstable spontaneous activity. In contrast, the great majority of adult cells were spontaneously active in the absence of stimulation, under similar conditions of urethane anaesthesia. 2. The distribution within cortical layers of cutaneously driven cells was similar in adult and 7 day old rats, and similar to that found in adult mammalian cortex by other workers. 3. 7 day old cells showed diminished excitability to cutaneous stimulation with stimuli at intervals below 10-15 sec, whereas adult cells could be successfully repetitively driven with stimuli at intervals of 500 msec. The low ability of the immature cells to follow repetitive cutaneous stimulation is not due to an overall depression of these cells excitability per se. Latencies of unitary responses in these immature cells were about sixfold those found in equivalent cells at maturity. 4. Columnar organization at seven days of age was similar in outline to that of the adult, but much less discrete. Receptive fields were considerably larger at 7 days and evidence is given that this may be due to inadequate surround inhibition. Immature vibrissae-driven units were directionally selective. 5. At 7 days of age, long inter-spike intervals were rare in spontaneously active cells with the result that inter-spike interval histogram distributions (i.h.s.) were approximately normal. Corresponding i.h.s. of adult cells invariably showed skew distributions. 6. Tactile stimulation of centre receptive fields produced an increase in 21 -2

5M. ARMSTRONG-JAMES 502 short and long intervals from spontaneously active cells at each age. In contrast to adult cells, the immature cells commonly responded cyclically, with alternating phases of increased and decreased firing rate for periods of up to 3 sec following punctate stimulation. 7. Decrease in spontaneous firing rate, following the first phase of excitation, was profound in 7 day old cells, and implied that inhibitory mechanisms operate at an early age in the rat somatosensory system. These mechanisms also appear to contribute to cyclical activity of 7 day old cells when driven by punctate cutaneous stimulation. INTRODUCTION

In recent years attention has been directed towards the functional development of evoked unitary activity in various areas of visual cortex of the cat (Hubel & Wiesel, 1963; Wiesel & Hubel, 1963; Barlow & Pettigrew, 1971), and a single paper exists upon columnar development shown by evoked unitary activity in somatosensory cortex of the kitten (Rubel, 1971). Numerous studies exist on the development of evoked potentials from a variety of cortical areas in immature mammals (for example, Scherrer & Oeconomos, 1954; Ellington & Wilcott, 1960; Grossman, 1955; Marty, 1962; Thairu, 1971). A recent study has also been carried out on the ontogenesis of evoked potentials together with some observations on unitary activity in the developing sheep foetus somatosensory cortex (Persson, 1973) (although this study has been limited to the trigeminal afferent inflow). There is therefore a paucity of data available for the functional development of the somatosensory system in particular, and for the ontogenesis of sensory activation of cortical units in general. The available evidence suggests that the cerebral cortex of the rat is at a very immature stage compared with other mammals at birth (Thairu, 1971; Armstrong-James, 1965) both with regard to its functional development and its histological synaptic status (Armstrong-James & Johnson, 1970; Johnson & Armstrong-James, 1970). The present investigation restricts itself to an examination of the functional status of cortical neurones at 7 days of age post-natal, the first age at which regular activation of S 1 cortical units could be obtained by natural stimulation of cutaneous areas. Several questions were posed for this investigation. Firstly, what evidence is there for columnar organization of S 1 primary somatosensory cortex, in early post-natal life? Secondly, how do receptive fields differ from those found in adult rat cortex? Thirdly what patterns of impulses are produced by immature primary somatosensory cortex units, in reply to natural stimulation? Comparisons are made with data available for mature mammalian primary somatosensory cortex (e.g. Mountcastle, 1957; Mountcastle, Davies & Berman, 1957; Welker, 1971) and with cortical

503 SOMATOSENSORY CORTEX OF NEONATAL RAT unitary activity evoked in species other than the rat at early stages of development. Further observations are concerned with a comparison of spontaneously active cells in adult and immature rat somatosensory cortex, and their responses to cutaneous stimuli. METHODS Sprague-Dawley rats were used in all experiments. Female adult rats were used exclusively, and 7 day old animals of both sexes. Litters were restricted to twelve at birth in order to obtain standard rates of growth. Rats were considered to be one day of age on the day of their birth, and adult at 180 g body weight. Animals were anaesthetized at both 7 days of age and at maturity with urethane (approx. 1 ml. 20% urethane in saline per 200 g body weight), via the i.P. route. Operations were carried out within 30 min of induction, and during experimentation anaesthesia was kept at a level whereby reflex withdrawal to squeezing of the hind foot was fairly brisk. In animals at both ages 1-4 sq. mm of cranium were removed overlying the presumed primary somatosensory cortex, S 1; the dura and pia were left intact. A specially constructed Perspex collar was mounted on the cranium, circumscribing the exposed cortex. This was cemented to the cranium with non-toxic oxyphosphate cement polymer (Durelon (R), ESPE GmbH) which set rigid within 5 min with no visible shrinkage. The collar contained a small thermistor (0.1 mm diameter) which rested on the edge of the exposed dura. Within the collar was an insulated heating coil. The cortex was covered with Agar-agar gel in saline 2-3 mm in depth which remained a gel at 37° C. This effectively dampened respiratory excursions of the underlying cortex. The gel was covered with 1 mm of mineral oil which prevented drying. The heating coil was connected to a DC power-unit whose output was controlled by the thermistor, such that the gel was maintained at 37 ± 0*2° C effectively keeping the cortex at this temperature. A similar system was used to maintain the body temperature. The thermistor in this case was rectal. The head was held rigidly by orientable clamps which located on the collar cemented to the cranium. Such a device was necessary in young animals in which the cranium was extremely soft; in practice the collar maintained the correct geometry of the brain extremely well.

Stimulation and evaluation of receptive fields For preliminary evaluation of receptive fields a variety of stimuli were used. These included puffs of air, movement of hairs, and the use of glass probes, von Frey hairs and nylon hairs. Once the receptive field was established an electromagnetic stimulator was used to operate a standard glass probe with a 0*2-0 5 mm spherical tip for the skin deformation, and a similar probe for hair displacement. The rise time of the mechanical pulse was usually maintained at 20 msec and this should be assumed unless otherwise stated in the text. This was the minimum rise time found for the system whereby overshoot was restricted to less than 5 % of the displacement. Displacements of the probe could be varied from 100 gr to 3 mm, although typically tactile receptors rarely required displacement in excess of 500 /tm. The total steadystate application period of the mechanical stimuli could be varied between 50 and 500 msec. Placement of the stimulus probe was carried out by means of rack and pinions coupled with micrometers for fine adjustment. Placements were normally carried out by observation under a dissecting microscope. For quantitative analysis of responses, displacements of skin were typically 50 % above threshold for centre

504

M. ARMSTRONG-JAMES

receptive field locations. All other locations within the receptive field were stimulated with identical pulses for determining the boundaries. At regular intervals the centre receptive field was tested to determine any change in the excitability of the neurone under investigation.

Recording techniques Glass micropipettes filled with 2 M-NaCl were used in nearly all experiments and recording was extracellular. A variety of micro-electrodes were tried in earlier experiments, including stainless-steel, tungsten and metal filled electrodes. However the glass micropipettes appeared to yield larger quantities of successful units. Tip diameters of these electrodes were 1 pzm or less, with resistances for successful electrodes being between 4 and 25 MQ. Signals were led directly into the input of an F.E.T. operational amplifier (Fenlow 5002), which had an input impedance of 400,000 MO, and a tested flat frequency response of 0-5 kHz, with the gain maintained 1-0. This miniature amplifier was carried on the probe which held the microelectrode, and shielding was therefore found to be minimally necessary. The current amplified signals were led into an AC amplifier constructed in the laboratory which had amplification capability of 1-10,000. Active filters were employed to 3dB cut-off above 5 kHz or 2 kHz as required, and below 5-150 Hz, also as conditions required. Signals were displayed on a Tetronix 502 A oscilloscope for visual monitoring, and the action potentials were simultaneously led into a variety of signal conditioning equipment. This equipment was all constructed in the laboratory and included a pulse height discriminator which produced logic pulses, and was capable of separating up to four groups of action potentials recorded at the same time into four discrete channels, on the basis of action potential height categories. These pulses were used for on line analysis of action potential data with a Biomac computer in later experiments. Other equipment included a gated frequency meter for estimating mean frequencies of firing at various intervals after stimulation or preceding stimulation, and electronic gates operated by the stimulus pulse to guide relevant portions of the response train of action potentials to the Biomac computer. Audio monitoring was employed throughout to set the pulse height discriminator level. All data were collected on FM tape, which included the stimulus, action potentials, logic pulses and speech. The speech channel also contained data facility which was employed from time to time.

Electrode advancement Apparatus for the control of the movement of the electrode was constructed in the laboratory together with the stereotaxic frame. The electrode was held in a probe which contained the first stage of amplification. The probe could be oriented in such a way that the electrode could be advanced at various desired angles into the cortex, which were allowed by the elevation of the collar mounted on the cranium. Electrode penetration angles up to 30 degrees away from normal were possible. Horizontal readings were in reference to Bregma, and corrected vertically in relation to the cortex surface with due allowance for the depth of the subarachnoid space. The cortical surface could usually be determined by an increase in the noise recorded by the system. Advancement of the probe was hydraulically controlled by ganged syringes filled with mineral oil recommended by Shell (Shell Tellus oil). The driving syringe was compressed against a powerful recoil spring by the use of a stepping motor. The motor rotated in reply to logic pulses, advancing 1/48th of a revolution per step, giving a final step advancement of the micro-electrode of 2-4 ptm per step, i.e. per pulse. Advancement rate could be varied from 1 step/10 see to 100 steps/sec. The control of the device was sometimes automatic whereby action

SOMATOSENSORY CORTEX OF NEONATAL RAT

505

potentials of a certain height were used in a feed-back mode which gated the stepping motor into the off position. On encountering a spontaneously active unit, therefore, electrode advancement automatically stopped. Forward and reverse one-step facilities were available for isolation of single units, to obtain maximum spike height. Backlash in the system was usually less than 1 /tm. Hiatological methods It was necessary to know the depth of the relative layers of the cortex at the adult stage and at 7 days of age and for this purpose a thorough investigation of three modifications of the Golgi method was carried out; these were the Golgi-mixed method, the Golgi Rapid method and the Golgi-Cox method. The latter gave the most statisfactory results for both adult and immature neurones, although there was some preference for pyramidal cell staining in layer V at birth and 7 days of age. Sholl's modification of the method (Sholl, 1953), with the toning technique using ammonia and gold chloride introduced by Da Fano (quoted in Bolles Lee, 1950) gave greater contrast of the neurones against background material. Sections were cut at 100 ,sm from celloidin embedded material, sections being normal to the surface of the cerebral cortex. Linear shrinkage of the material was estimated from electrode lesions at a known depth below the cortex. This shrinkage was approximately 15 %. Depths of cortical layers were therefore corrected from this estimation. RESULTS

Cortical depth and depth of layer The cortical depth was estimated at the anterior region of the forearm area of the somatosensory cortex, from celloidin sections of Golgi-Cox stained material (see Methods). The position of the deepest neuronal cell body was used to determine the transition point into white matter. The depth of the first layer, the plexiform layer, was estimated by averaging the depths of the bouquet cells, the most superficial cells of the cerebral cortex, or cajal-retzius cells if they were present (the latter were rare at young ages and appeared to be absent at maturity). Layers II and III could not be separately distinguished at any age in the rats studied. They are commonly called the outer granular layer in other mammals (e.g. Sholl, 1953), but in this investigation no basis for subdivision could be made. The layer was therefore termed layer II/III, and predominated in large numbers of small pyramidal cells with most of their apical dendrites penetrating layer I at all ages after birth. Layer IV, the inner granular layer of stellate cell layer, was easily distinguishable from other layers due to the presence of excessive numbers of stellate cells at all ages from 4 days of age onwards. Layer V was equally easy to distinguish by the presence of large pyramidal cells and layer VI was determined from the presence of cells with little orientation of their rather long poorly branching dendrites at all ages. Judgements on the depths of all layers was determined by a change-over in predominant frequencies of cell types with depth, and the results are given in Table 1.

M. ARMSTRONG-JAMES

506

Classification and distribution of units: spontaneously active and silent cells A total of 161 penetrations throughout the depth of the somatosensory cortex (S 1) in 7 day old animals were made, involving some seventy-two successful preparations. Eight hundred and seventeen cells were encountered which were silent in the absence of international stimulation or spontaneously active, also in the absence of intentional stimulation. Spontaneously active cells could be classified further into two main types. TABix 1. Depths of cortical layers and main types of cells found Depth (gim) , A

Cortical layer Layer I Layer II/III Layer IV Layer V Layer VI

Adult 0-218 218-595

A

7 days

0-103 103-495

Predominant cell type None Small pyramidal/ bouquet Stellate Large pyramidal Polymorphous

495-897 897-1270 1270-1840 The s.E. of the mean depth was less than 8 % in all cases. 595-1265 1265-1668 1668-2305

(1) Those which were quite clearly injured, and showed all the well known features of injury discharge. These cells invariably 'died' within a few seconds of being encountered, presumably due to impalement by the micro-electrode of some part of their membranes; (2) 'Stable' spontaneously active cells which showed no overt signs of injury. The criteria for stability of cells are defined as follows. In the absence of stimulation the mean firing rate must exceed 1/sec over consecutive 1 min periods. This definition is, of course, arbitrary, although cells which fired at lower rates did so at average frequencies below 0.2/sec in an irregular manner. Secondly, cells showing injury discharges or other signs of deterioration such as decreased spike height or abrupt alteration in firing rate were labelled 'unstable'. The numbers of spontaneously active cells encountered were exceedingly few. About 12 % of all cells showed some sort of spontaneous activity, although the great majority of these were unstable in one way or another. Only some 3 % of all cells could be classified as stable spontaneously active cells which could be influenced by cutaneous stimulation. Since 88 % of responsive cells were silent in the absence of peripheral stimulation, the electrode had to be advanced very slowly through the cortex with continuous assessment for unitary evoked activity. Such a procedure was very time consuming and was aggravated by the long intervals between stimuli

SOMATOSENSORY CORTEX OF NEONATAL RAT 507 required to give adequate activation of units (see below, The effect of stimulus repetition rate on responses of 'silent cells'). As a consequence of this, it was only possible to produce results from a maximum of five penetrations of cortex in most preparations. Cortical distribution of units. In sixteen of the experiments at 7 days of age, at least one column within each experiment yielded five or more spontaneosly active or driven silent cells. At 7 days of age, a detailed analysis of all these 'high yield' columns was carried out for distribution of units within the cortical depth, according to the layers previously determined (see Table 1). The depth of each active cell was registered on the microdrive unit and its position corrected for the angle of penetration. The results from some 309 cells were pooled into two categories: (a) spontaneously active, and (b) solent cells. The results for 292 silent but driven cells are shown in Fig. 1. Although spontaneously active cells showed a similar distribution within the cortex to silent cells, too few were found to be certain of this finding. The greatest number of driven silent cells, therefore, occurs within the inner stellate layer, layer IV (41.7 % of silent cells). Approximately equal numbers of these cells were found in the small and large pyramidal cell layers (25.3 and 29-7 % respectively). Within the experimental conditions used in the present investigation it would appear that the deepest layer of the cortex, the polymorph layer, is not strongly involved in the reception of tactile information at 7 days of age.

Effect of stimulus repetition rate on responses of 'silent cells' With mechanical stimulation of the skin it has previously been reported that the stimulus repetition rate must be very low to obtain consistent activation of immature cells (Armstrong-James, 1973). Stimulation rates exceeding 1/10 see often led to a decrease in the number of action potentials produced by silent cells as the investigation proceeded. Fig. 2 shows the post-stimulus histograms generated by a typical cell in response to centre receptive field stimulation. Stimulation of lateral palm at 1/5 see (A) yielded an average of 3-3 action potentials per stimulus, whereas with stimulation at once per 15 see (B) maximal activation occurred at 6-4 spikes per stimulus. The response pattern for B was better synchronized to the stimulus than at the higher rate of stimulation in A. Such a finding was typical for silent cells. Preliminary investigations of this type had to be carried out with every cell investigated in earlier experiments until it became clear that the great majority of 7 day old S 1 cortical cells would respond consistently only at stimulation rates of 4 times a minute or below.

M. ARMSTRONG-JAMES

508

Columnar organization at 7 days of age: topographical representation At 7 days of age evidence was found for strong vertical columnar organization of S 1 cortex in a manner similar to that seen in adult feline visual cortex (Hubel & Wiesel, 1963), adult feline somatosensory cortex, (Mountcastle, 1957) and for adult rat vibrissae somatosensory cortex (Welker, 1971). One method of determining the degree of columnar 40

30

20U

10

0_

11/111

IV Cortical layer

_

V

VI

Fig. 1. Distribution of 292 'silent' cells responding to tactile stimulation, in the somatosensory cortex of the 7 day old rat. The distribution is shown with respect to layers of the cortex evaluated at this age. Further details in the text.

organization is to use oblique penetrations of cortex, and this technique was often employed in the present investigation, although the great majority of penetrations were at right angles to the cortical surface. The receptive fields of cells encountered in an oblique penetration of cortex are shown in Fig. 3. The first few cells encountered within superficial cortex were predominantly responsive to punctate mechanical stimulation of the plantar palmer surface, whereas the remaining six cells in the lower

SOMATOSENSORY CORTEX OF NEONATAL RAT 509 part of the cortex predominantly responded to stimulation of the plantar surface of the little finger. Clearly the electrode has passed across the junction of two columns, one representing cells responsive to the palm, the other to the little finger. The fact that this separation of columns is well defined can be seen when the relative numbers of action potentials generated by cells are compared from stimulation of the two sites. The change-over to little finger dominance occurs quite clearly between units D and E, the separation of these units being less than 100 jum. Such a clear change-over is fortuitous of course, since the micro-electrode does not always pass cleanly across the junction of two columns. 0.5_

A

|

.i I

.

05 0

Time (sec)

Fig. 2. Post-stimulus histograms produced for a single cell in 7 day old somatosensory cortex. In A the response to ten stimuli at intervals of 5 sec is shown. In B the response of the cell to ten stimuli at intervals of 15 sec is shown. In each case the centre of the receptive field was stimulated with identical pulses of 180 msec duration. The numbers of action potentials produced per stimulus at the higher rate (A) was 3-3 spikes per stimulus, whereas stimulation at the lower rate (B) yielded 6-4 spikes per stimulus (B). Further decrease of the stimulus rate failed to give a greater response function (not shown). Note the greater coherence in the response pattern of B compared with A.

Separation into columns is most clearly seen in distal forelimb areas and within the vibrissae fields, and seen at its most ill-defined within cortical areas responding to flank stimulation. In common with this finding, receptive fields of columns of cells responding to stimulation of cutaneous areas of the extremities and vibrissae, are very much smaller than those responding to stimulation of areas proximal to the vertebral column. This phenomenon is illustrated in Figs. 4 and 5, in which the mean

510 M. ARMSTRONG-JAMES receptive fields for cells from several adjacent vertical of cortex are shown. In Fig. 4 it can be seen that the more proximal the location of the receptive field within the forearm, the larger is the total receptive field for the respective column activated. Secondly, in comparison with Fig. 5, in which mean receptive fields for flank columns are evaluated, it is evident :E or

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L

BE C

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I

G~~~~~~~ 1600 pm Fig. 3. Shift of dominance in receptive fields of cells encountered in an oblique penetration of the forearm area of somatosensory cortex of a 7-dayold rat. Cells encountered within the penetration are shown as black dots in the electrode trajectory shown in the centre of the Figure. To the right are shown the receptive fields of the individual cells on diagrammatic figurines of the rat forelimb. Centre receptive fields are shown as black areas with excitatory surrounds as hatched and stippled areas. To the left are shown the mean numbers of action potentials in reply to 7-20 stimuli applied to the little finger (A) and adjacent palmar area (P), for each cell encountered. Scale (top left): Mean nos. of action potential/stimulus. All units replied to the early part of the stimulus only, the stimulus lasting 180 msec.

that the latter have very much larger receptive fields. Total fields sometimes involved the whole of the flank and belly on one side (Fig. 5, column B). The degree of overlap of representation in adjacent columns was also always remarkable for those columns driven by flank stimulation. If a column is taken to be determined by its centre receptive field location, some estimation of the lateral separation of forelimb columns can further be deduced from experiments such as those illustrated in

SOMATOSENSORY CORTEX OF NEONATAL RAT 511 Fig. 4. This experiment was typical in as much as four discrete maximal receptive fields namely from two adjacent digits, the palm and the wrist, activated four independent columns, maximally separated from one another by about 1 mm. Findings with regard to hind limb columns were similar, although the columnar organization of hind limb activated cortical areas was not so discrete (compare Figs. 4 and 8, 7-day-old animals). Total receptive fields of cells responding to hind limb stimulation were also a good deal larger than their forelimb counterparts.

E

A~~~~~~~~~

Ant j.A

B- *C D

Fig. 4. Mean receptive fields for cells encountered in adjacent vertical penetrations of 7 day old rat somatosensory cortex. b, location of Bregma arrow indicates rostral direction. Horizontal and vertical scales are in mm. Between four and eight cells were encountered in each column. In all cells, centre receptive fields overlapped for individual columns. Distal fields are smaller than proximal fields. Hatched areas show excitatory surrounds, with the limits of the excitatory surrounds being indicated by the stippled areas. Upper scale refers to figurines.

From all the columns of cells investigated, the vibrissae produced the smallest receptive fields. Units with fields involving only two vibrissae were quite commonly found, although other cells responded to movement of all the vibrissae. An example of a typical electrode trajectory with total receptive fields of the cells encountered is shown in Figs. 6. A further

512 M. ARMSTRONG-JAMES finding was the profound directional selectivity that vibrissae driven units exhibited, a feature which is discussed later in this paper. On the basis of the types of experiments outlined above, centre receptive field representations of cutaneous areas were evaluated for each of fiftyeight columns in fourteen 7 day old animals and their topographical localization on the cortex surface noted. Fig. 7 shows the pooled results from these experiments. Somatotopic projection when compared between 60mm

l l~ ~ ~ ~ ~ 9

IC) Ant. 1

2

4 mm

3

B

A

Fig. 5. Mean receptive fields for cells encountered in adjacent vertical penetrations of somatosensory cortex driven by stimulation of neck, thorax and belly. Sizes of total receptive fields for columns should be compared with Fig. 4. Areas involving the main trunk of the animal project to very small volumes of cortex. Other details as for Fig. 4; 7 day old rat.

SOMATOSENSORY CORTEX OF NEONATAL RAT 513 different preparations was highly variable, although within individual experiments a logical format of representation was apparent. Pooled experiments indicate that forelimb representation is anterior and medial to the mid line just behind the level of Bregma, whereas hind limb representation is more posterior and medial to the mid line. Vibrissae fields

Fig. 6. Receptive fields of cells in a vertical trajectory encountering cells only driven by movement of the vibrissae; 7 day old rat. Other details as for Fig. 3. All cells encountered responded preferentially to upwards movement of the vibrissae. Vibrissae which failed to drive cells are shown as interrupted lines.

were found mainly lateral to the forelimb areas whereas flank, belly and shoulder columns were found between the fore and hind limb areas, with a tendency to be lateral to them. Tail columns (two only) were found medial to the great majority of hind limb columns and somewhat posterior to them also. Within individual experiments forelimb columns were such

M. ARMSTRONG-JAMES 514 that digit columns were located more anterior and medial than palmar and wrist columns (Fig. 4). The results also clearly indicated that greater volumes of cortex are devoted to more distal regions of the limbs and vibrissae. Although total fields for the vibrissae were not possible to evaluate in the individual mm

1

2

from mid line

4

3

I

l

I

5

1-

+0

F V F F H V V F H F F F F F V F F F F F S S FF D V V V e seS FF V V S @ S

Br-

1-

E E

2-

F

F

(E)

(E)

F~~~(9

(e}

34 44

V V

Fig. 7. Topographical locations (pooled for fourteen experiments) of vertical columns in the 7 day old cortex. The location of Bregma is shown as Br. Rostro-caudal and lateral scales are in mm, the lateral scale being corrected for the curvature of the cortex. Posterior portions of the body are ringed. The explanation of symbols is as follows: F, forelimb, P, hindlimb, H, regions of the head other than vibrissae V, vibrissae, D, dorsal trunk, S. anterior flank and thorax, R, posterior flank and abdomen, T, tail. S and R exclude dorsal areas. Further details in the text.

experiments, it appears that they have the greatest amount of cortical space devoted to them. The forelimb projects to the second greatest volume of cortex, followed by the hind limbs, anterior and posterior flank, belly, dorsum and tail. It was not generally possible to determine the

SOMATOSENSORY CORTEX OF NEONATAL RAT 515 projection of the remainder of the head due to obstruction by the stereotaxic apparatus. Differences in receptive fields at 7 days and maturity In all cases the sizes of receptive fields, including the excitatory surrounds, were considerably larger at 7 days of age than those found in the adult animal. For example, in adult animals, receptive fields involving the whole of the trunk on one side were never found for cells in primary somatosensory cortex, whereas they often were at 7 days of age. With regard to fore and hind limb areas, receptive fields could very often be found for individual digits with weak excitatory and occasionally inhibitory surrounds at maturity. At 7 days of age, on the other hand, fields where one might expect inhibitory surrounds at maturity were replaced by moderate or weak excitatory surrounds. Fig. 8 shows a comparison between receptive fields of vertical columns of cells driven by hind leg stimulation at the two ages. These columns are typical of those found at the two age groups. The centre receptive field fluctuates somewhat randomly between ankle, palm and digit areas in the hind limb vertical trajectory at 7 days of age, the column affinity being predominantly palmar and digit. This pattern was generally the rule at seven days of age for hind limb columns, although somewhat better or worse column affinities than this were found. Centre receptive fields involving only the digits were never found for hind limb driven columns at 7 days of age, although the occasional unit did show a centre receptive field located to one digit. In all distal hind limb driven cells at this age, the excitatory surround always involved the palmar area for individual cells at the very least, and very often involved areas up to and including the ankle. On the other hand, at maturity a very small total receptive field was generally the rule for distal hind limb driven units. For those trajectories predominantly involving the digits, the centre receptive field could be traced to one segment of the digit only on many occasions, with weak excitatory surrounds sometimes only involving the same digit (Fig. 8). Centre receptive fields involving the hind limb digits at maturity rarely had excitatory surrounds extending beyond the palm and often did not involve the palm. These qualitative differences between the two age groups were similar for forelimb activated units, although the column affinity of different cells in the column for the same receptive fields was greater at 7 days of age for forelimb than hind limb columns.

Latency of response to tactile stimulation: forepaw units Latency histograms were constructed for some forty-one 7 day old cells responding to brief suprathreshold mechanical stimuli applied to glabrous

516 M. ARMSTRONG-JAMES skin forepaw centre receptive fields. The average latency was evaluated for each unit and the distribution of these average latencies is shown as a histogram in Fig. 9. There is a very considerable scatter in the latencies of these units, and there was a loose inverse relationship between the mean number of action potentials produced by the cell and the latency. The mean latency for all units investigated was 88+ 48 1 msec (S.D.). This latency is very considerably greater than for comparable adult forepaw latencies of silent cells which were estimated in several experiments on fifteen forepaw units at a mean latency of 13-8 msec + 4 3 msec under similar conditions.

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7 1I 2

a b

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5--h

5,

6

6'

d

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a

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W-. 1600 jam

74

- 1800

7 days

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Fig. 8. Proximal hind limb receptive fields of cells in typical vertical trajectories of 7 day (left) and adult (right) somato-sensory cortex (S 1). Details of nomenclature and layout as for Fig. 2. V represent locations which when stimulated caused decrease in spontaneous firing rate of the unit under investigation. 0 represent areas causing no change in firing rate. Hatched/stippled areas: excitatory surrounds. Black areas: centre receptive fields.

Patterns of responses from 'silent' 7 day old cells A considerable variety of patterns of activation of silent cells occurs at 7 days of age, largely regardless of the site of cutaneous stimulation. For example, 'on', 'off' and 'on-off' types of responses were detected for

517 SOMATOSENSORY CORTEX OF NEONATAL RAT receptive field stimulation of glabrous skin, potentially hairy skin, vibrissae and sinus hairs. One difficulty encountered during these investigations was the extraordinary latency of some of the responses (Fig. 9) and in the consideration of this finding it was necessary to use extremely long duration stimuli. By doing so only then was it possible to determine whether a cell responded to the beginning, the steady application, or the termination of the stimulus. Fig. 10 illustrates this point in principle, in a 12

0

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88 -

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E

z 0 -*

0

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I

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100 200 Mean latency (msec)

I

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300

Fig. 9. Seven days of age. Mean latencies of response to tactile stimulation of 'silent' forepaw units. Histograms constructed for forty-one cells whose centre receptive field was stimulated with stimuli with a 10 msec rise time. Latencies were evaluated from 5 msec after the stimulus had commenced, and are therefore subject to an error of + 5 msec.

typical experiment in which stimuli of two durations were used. For the first cell encountered (590 Itm) post-stimulus histograms were constructed using 180 msec stimuli ( x 10). The response gives the appearance of being an 'off response'. Using longer stimuli (400 msec) illustrates that this is not the case. The latency of the response remains the same and is quite clearly related to the onset of the stimulus. The post-stimulus time histograms (p.s.t.h.s) from other cells encountered within this trajectory serve to illustrate other common features of these immature cells. First, there is some variation in latency from cell to cell, using identical stimuli applied to the centre receptive field, and secondly, the pattern of responses is not always simple. For example, the last cell encountered has a three component p.s.t.h.s whose pattern is evidently not related in a simple manner to the dynamic and static portions of the stimulus.

518 M. ARMSTRONG-JAMES The response patterns of cells encountered within individual trajectories were sometimes almost identical, most commonly so with columns activated by the vibrissae. Fig. 11 illustrates p.s.t.h.s constructed from three cells encountered within a single trajectory in which the p.s.t.h.s. forthe

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10 0-5 Time (sec) Fig. 10. Forepaw units, 7 days of age. Post-stimulus histograms constructed for individual cells at various depths in a vertical trajectory. Centre receptive fields were similar for each cell. A 1 and A2 are responses from the same cell, A 1 being in response to stimuli of 180 msec duration, A 2 in response to stimuli of 400 msec duration. Cells B, C and D were found at successively greater depths in the trajectory, all of them responding in part to the onset of the stimulus. Further details in the text. The stimulus is shown as a heavy black line under each p.s.t.h. Figures to right: depth in um of cell.

519 SOMATOSENSORY CORTEX OF NEONATAL RAT different cells are remarkably similar. Each cell was solely activated by a common group of posterior vibrissae.

Directional selectivity of vibrissae units Vibrissae units were also usually highly selective about the direction of deflexion of the appropriate vibrissae causing their activation. A variety of orientations appeared to be most adequate for different columns, and most commonly units were preferentially driven by movement in one 1-

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1-0 05 Time (sec) Fig. 11. P.s.t.h.s constructed in response to 400 msec stimuli for three 7 day old units encountered successively within a vertical trajectory of cortex. All cells were driven only by the posterior group of vibrissae. Note the similarity in the peaks of the histograms related to the start and end of the stimulus. Stimulus application is shown as a continuous line under each histogram, upward deflecting vibrissae occurring at the onset, downward on 0

termination of the stimulus.

quadrant of direction only. Figs. 11 and 12 show responses from two columns of cells, one of which was highly directionally selective with regard to the vibrissae that drove it (Fig. 12). The other column (Fig. 11) was not so directionally selective but clearly produced more consistent

M. ARMSTRONG-JAMES 520 responses upon upward movement of the whiskers. (It should be noted that the rise time of the stimulus (on) was the same as the fall time, namely 20 msec). Some forty-five vibrissae units were studied adequately,

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Fig. 12. Seven day old cells. Vibrissae driven units. Details as for Fig. 12 and 13. All cells encountered in this vertical trajectory responded only to the 'off' portion of the stimulus. Evidence for this is shown in the two histograms generated from the same cell (at 645 gm), where stimulus durations were different. The peak of the p.s.t.h.s are related to the off portion of the stimulus (forward movement of vibrissae). Further explanation in the text.

and in only nine of these was it not possible to show some degree of directional selectivity. It cannot be excluded that more adequately controlled experiments on these remaining nine units may have shown some

521 SOMATOSENSORY CORTEX OF NEONATAL RAT degree of directional selectivity. The one factor that was always apparent for vibrissae columns was that each unit within the same column nearly always gave very similar average response histograms, although cells within central regions of cortical depth tended to give greater average numbers of action potentials per stimulus.

Variations in patterns of response in columns On other occasions two or more types of response patterns were elicited from different cells within the same column. Fig. 13 shows an example of p.s.t.h.s recorded from successive cells in a column activated by stimulation of the posterior flank. The first two cells encountered responded to the onset of the stimulus with a rather ragged latency, but nonetheless giving a well defined onset p.s.t.h. and this response was followed by irregular activity lasting on occasions 500-600 msec after termination of the stimulus. Cells encountered further down this column deeper within the cortex produced well defined histograms consisting of 'on' and 'off' portions with some tonic activity during stimulus application, and no after-burst. Sustained post-activation facilitation for periods of 500 msec or more were fairly common; another example can be seen in Fig. 12 at 1325 gtm cortical depth from a vibrissa driven unit. The very sharply defined grouping of impulse patterns that were so common from vibrissae units were not by any means restricted to them. Fig. 14 shows p.s.t.h.s constructed for a column of cells driven by tactile stimulation of the shoulder. As far as could be judged these cells were not activated by the very small hairs which were found on the animal at this stage of development, since deflexion of them did not cause discharges of the cells encountered. The response patterns are very similar to those that would be expected from vibrissae driven units, and it seems probable that a similar type of dynamic receptor may be involved. Similar patterns of impulses in response to the dynamic components of the stimulus were obtained from a variety of sites, including sinus hairs and fore and hind paw areas. In common with vibrissae columns, successive cells in these columns tended to exhibit similar p.s.t.h.s. In general, therefore, within vertical penetrations of cortex, similar patterns of activation of successive cells were far more consistently obtained from those columns activated by the vibrissae than from any other cutaneous area. This finding was similar for occasional columns of cells activated preferentially by the dynamic portion of stimuli applied to other sites. There was little clear indication of sustained activity in direct relationship to punctuate cutaneous stimuli that might indicate 'tonic' activation

M. ARMSTRONG-JAMES 522 of normally silent cortical cells, at 7 days of age, even though many cells produced after-bursts of activity.

Patterns of response from spontaneously active 7 day old cells Stable spontaneously active cells are exceedingly rare at 7 days of age (Armstrong-James, 1973). Most of those that were encountered showed some sign of deterioration and only twenty-three cells were found which 05 _

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SOMATOSENSORY CORTEX OF NEONATAL RAT 523 could be termed 'stable' in their activity (see p. 523 for criteria on stability of spontaneous activity). Characteristically these 'stable' cells either exhibited a very steady firing rate or showed small (10-20 %) variations in firing rate with a periodicity of 20 sec or more with no change in spike height. The latter were in the minority and no explanation can be given for their behaviour. 05

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Fig. 14. P.s.t.h.s of 7 day-old units successively encountered in a vertical trajectory in which all cells exhibited centre receptive fields on the shoulder. Note the similarity to p.s.t.h.s generated by vibrissa driven cells (Fig. 13). Further explanation in the text. Other details as for Fig. 12.

Most spontaneously active cells responded to cutaneous stimulation and during periods when no stimulation was being given their stability was tested by constructing interval histograms. The effect of slight injury by electrode movement on such cells is shown in Fig. 15. In general, the mean interval decreases on damage, with a marked increase in long intervals resulting in histograms with a greater degree of skewness than the healthy stable cell. Interval histogram analysis was carried out on twenty-three stable spontaneously active seven day old cells. Fourteen of these cells were well driven by cutaneous stimulation and their responses to centre receptive field suprathreshold stimuli analysed as follows: Interval histogram analysis was carried out for twenty-three spontaneously active 7 day old cells. For the fourteen 7 day old cells which

M. ARMSTRONG-JAMES 524 showed responses to cutaneous stimulation, interval histograms were constructed from successive 3 second periods immediately following punctate stimulation, by use of electronic gates (see Methods). Centre receptive fields were stimulated for all the inter-spike interval histogram distributions described below, using 50 % above threshold 100-150 msec stimuli. Similar data were produced for spontaneously active adult cells.

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Firing patterns of spontaneously active cells: without stimulation. A noticeable feature of immature cells was the rarity of bursts of impulses, with long intervals between the bursts, that were common in spontaneously active adult cells. Such differences between the two age groups may be clearly seen from a comparison of the most common types of interval histograms constructed for adult and 7 day old cells (Fig. 16). In general, it is evident that adult cells produce far greater numbers of not only long inter-spike intervals, but also short intervals as well. Such a finding indicates a fundamental difference in the patterns of 'spontaneous' activation of the cells of the two age groups. Undriven 7 day old cells were very rarely

SOMATOSENSORY CORTEX OF NEONATAL RAT 525 able to produce intervals shorter than about 20 msec, whereas adult cells commonly produced intervals of 5 msec or less without peripheral stimulation. On the other hand relatively larger numbers of intervals exceeding 400 msec occur from spontaneously active adult cells when compared with 7 day old cells. A few adult cells failed to show any long intervals, and thus exhibited a short-tailed as opposed to a long-tailed skew distribution Adult

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of interval categories. More commonly the tendency for a proportion of adult cells to fire in bursts of short intervals with long intervals between bursts was apparent (Fig. 16, cell C). In Fig. 17 mean and modal intervals are compared at the two ages. An examination of the distribution of the modal intervals of spontaneously active cells at each age emphasizes the difference in the fundamental firing patterns between mature and immature cortical cells. The most common modal interval was about 90 msec at 7 days, and about 20 msec at maturity. There was also a fourfold difference in the mean modal intervals of these cells in the two age groups. What is also apparent from Fig. 17 is that there is a much greater difference between mean and modal intervals at maturity when compared

M. ARMSTRONG-JAMES with 7 day old cells. Secondly, mean intervals at the two ages cover a rather similar spectrum of intervals. This indicates that the mean firing rate of cells at each age group is rather similar, although modal intervals at maturity are only a fraction of those at 7 days of age. Over-all these findings confirm that adult cells tend to fire phasically during spontaneous activity with intervals of varying duration between bursts, whereas 7 day old cells tend to fire tonically. 526

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It should be noted that adult cells occasionally fired in 'doublets' or 'triplets' with very short (1-5-2 msec) intervals between successive spikes, these very short bursts occurring at fairly regular intervals. Such a pattern of firing always gave dual peaks to the interval histogram within the short 0-80 msec interval range. This pattern of firing was never observed in the present investigation of 7 day old cortical cells. These types of cells were excluded from the comparative analysis of interval histogram distributions for the two age groups.

Effect of cutaneous stimulation on inter-spike intervals. Activation of S 1 cells with standard 120 msec, just suprathreshold indentations at the

527 SOMATOSENSORY CORTEX O NEONATAL RAT centre of the receptive field, showed some interesting points in common for the two age groups, when the modal intervals were compared. The results are shown in Fig. 18. Modes of 7 day old cells, in the spontaneously active state, ranged from 38 to 175 msec, with an average mode of 102 msec. When these cells were driven by cutaneous stimulation, the mode shortened to an average of 15 10

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54 msec (range 22-75 msec). Although at maturity modal intervals were about one quarter of those in 7 day old cells, the pattern of change of mode was extremely similar in both age groups (Fig. 18). Adult cells had an average modal value of 18-4 msec (range 4 8-90 msec) in the spontaneous active state, and 8-65 msec (range 4-8-12-4 msec) in the peripherally driven state. The shortening of the mode due to cutaneous stimulation is therefore to an average of 56 % of the control at seven days, and to 57 % of the control at maturity.

528 M. ARMSTRONG-JAMES Fig. 18 also shows some data collected from some twenty-six stable 7 day old cells which were normally 'silent', i.e. exhibited no spontaneous activity, and were only active when driven by cutaneous stimuli. Their range of modal values was greater than those for similarly driven spontaneously active immature cells, although the average mode was similar (49.6 msec compared with 54 msec). The implications from this observation are not entirely clear, although it seems that the modal value in the driven state is not simply dependent on the level of spontaneous activity at 7 days of age. R 20

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A shortening of the modal value of the interval histogram during cutaneous stimulation clearly indicates excitation of the unit under investigation. The previous data have shown that rather similar mechanisms of excitation exist for adult and 7 day old cells, although the shorter intervals are about four times as long at 7 days compared with maturity. In comparing the driven and the undriven state, interval analysis, using the 2000-5000 msec gate after each stimulus, should show any change in

long intervals also. Fig. 19 shows an example of a cell from 7 day old cortex in which cutaneous stimulation produced an increase in both long and short

SOMATOSENSORY CORTEX OF NEONATAL RAT 529 intervals, with a decrease in intermediate interval durations, compared with the control. In this situation there is clearly a mixture of excitation and depression in spontaneous activity going on, although such an analysis does not show the sequential nature of the two processes. The latter can of course be shown by average response post-stimulus time histograms

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Fig. 20 shows an i.h. so constructed for a 7 day old cell in response to glabrous skin stimulation using standard stimuli, together with an i.h. analysed for the equivalent time period in the undriven, spontaneously active state. Below is the p.s.t.h. for the driven state. The i.h. shows a small decrease in modal value when the cell is stimulated, and an increase in the numbers of long intervals (R in the i.h.d.). Longer intervals are clearly contributed by a post-excitatory decrease in spontaneous firing rate which shows in the p.s.t.h. and appears to last about 500 msec in this

M. ARMSTRONG-JAMES 530 case. This 7 day old cell may also show 'rebound excitation', which is also sometimes found with naturally activated mature cortical cells.

Cutaneously evoked cyclical activity Post-stimulus time histograms exceeding 800 msec were constructed for 18 spontaneously active adult cells and eight spontaneously active 7 day old cells activated by glabrous skin in the pads of the fore and hind limb. Centre receptive field stimulation was carried out with standard stimuli

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SOMATOSENSORY CORTEX OF NEONATAL RAT

531 lasting 120 msec with a rise and fall time of 20 msec. Detection of changes in mean firing rate in comparison with spontaneous activity is facilitated by conversion of p.s.t.h.s into cumulative frequency plots. Fig. 21 shows such plots derived from p.s.t.h.s generated by 50-100 stimuli to centre receptive fields. These plots are typical of those produced by adult cells. In Fig. 21 A, B, and C, dotted or straight lines show the plots for spontaneous activity (control): filled dots show the plots in reply to cutaneous stimulation. Where the slope of the plot is less than the control, inhibition of spontaneous activity must be assumed to have occurred. Conversely where the slope is greater than the control, excitation is presumed to have occurred. Decrease in spontaneous activity following peripheral stimulation can of course arise by local inhibition via interneurones, or by disfacilitation whereby on-going influence of excitatory cortico-petal afferents is removed by inhibition of their sources. No distinction is drawn here, and for the purposes of brevity the term 'inhibition' is used to cover both eventualities.

With cutaneous stimulation, nearly all adult cells showed primary phases of excitation to a varying degree, followed by inhibition of spontaneous activity (Fig. 21 A, B, and C). Occasionally, however, two or more cycles of excitation and inhibition could be detected (Fig. 21 D). In contrast to adults cyclical activity in the immature cortex was a prominent feature of responses to stimulation of cutaneous centre receptive fields. Evidence of its time course and nature are shown in the cumulative frequency plots shown in Fig. 22. Periodicities in firing rate were sometimes detectable for up to three seconds after a single stimulus. In common with adult cells the primary phase of excitation (E 1) was always followed by an inhibitory phase (I 1). Five out of the eight immature cells studied in detail showed additional phases of excitation and inhibition following I 1. Four of these cells are shown in Fig. 22 (B, D, E and F). Periodicities were different in different cells, but were of the order of 300-800 msec in the few examples found. In all of the eight cells investigated, recovery to control levels occurred within 3-4 sec of the cessation of the stimulus. In the eighteen cells from adult cortex studied in detail, four showed a secondary phase of excitation in response to centre receptive field stimulation, and two of these also showed a secondary phase of inhibition below control levels. Estimation of E 1 and I 1 was carried out from cumulative frequency plots. Data were taken from eight immature and eighteen adult cells. The peak of E 1 and Ii were found by measuring the slope of the cumulative frequency plots where the slope was greatest and least accordingly 22

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SOMATOSENSORY CORTEX OF NEONATAL RAT 533 (e.g. Fig. 21 D, lines a and b). These slopes were calculated from 100 msec of counts in each plot. Durations of E 1 and I 1 were also measured. It was found that the peak of E 1 (first phase of excitation) in response to centre receptive field stimulation, was about twice as great in the adults as at 7 days of age (515 % ± 132 % (S.E. of mean) of the spontaneous rate in the adult; 228 + 32 % at 7 days). The peak level of inhibition of spontaneous activity (Ii) following E 1 was similar at the two ages studied (57 + 5-5 % below control for adult; 65 + 11*4 % at 7 days). However, this period of inhibition continued for a greater period of time at 7 days of age than at maturity lasting about twice as long in the immature cells as in the mature cells (905 + 83 msec (S.E. of mean) at 7 days; 487 + 73 msec adult). DISCUSSION

Columnar organization of immature cortex The experiments reported here have shown that both somatotopic and columnar organization were relatively well developed in 7 day old S 1 cortex. Certainly some powerful system for insulation of adjacent columns from one another must exist at an early age since the present study has shown sharp boundaries between neighbouring vertical columns of cells, each column having definitively different centre receptive fields. No direct evidence for lateral inhibition was found although intracellular recordings would probably be necessary to confirm its absence. Caley & Maxwell (1968) have commented on the appearance of columns of cells in their histological investigation of immature cortex, as has Rubel (1971) for somatosensory cortex of the kitten. However, the width of these workers columns are an order of magnitude less than the apparent width of functional columns found in the present study. The vertical alignment of cells may, however, aid in producing the functional columns found in the present study. With regard to 7 day old S I topographical arrangement, the relative positions of somatotopically organized columns compares favourably with the projection found in the adult (Woolsey & Le Messurier, 1948; Angel, 1967; Carter, Holmes & Houchin, 1969; Welker, 1971). Furthermore the relative amount of cortex devoted to handling information from particular areas of skin appears to be similar at the two ages. Since 7 days of age was the earliest time at which responses to cutaneous stimulation could be recorded, it would seem that the main anatomical substrates for columnar organization are present before the system becomes functional.

Sizes of receptive fields The findings presented here on large receptive fields in immature compared with adult S 1 cortical cells have not previously been reported. Rubel 22-2

3M. ARMSTRONG-JAMES (1971) in his study on new born-kitten S 1 cortex stated that receptive fields were virtually identical to those in the adult. However, it appears that he determined the extent of the centre receptive field only, without an investigation of surrounds. Hubel & Wiesel (1963) in a study on nine cells at 8 days of age and eight cells at 14 days of age in kitten striate cortex found that receptive fields were similar to adults also. It seems possible their subjects were already too mature to show differences with the adult although their observations have recently been challenged by Barlow & Pettigrew (1971). In consideration of receptive field size, at seven days of age no cells were found which responded solely to movement of a single vibrissa. The minimum receptive field normally was from about six vibrissae, and occasional cells were driven by nearly all of the vibrissae as far as could be judged. This is in sharp contrast to the adult in which individual columns of cells respond to movement of single vibrissae only (Welker, 1971). Small receptive fields of S 1 cortical cells may be due to a number of reasons, one of the most favoured being the mechanism of lateral or surround inhibition effectively reducing the size of receptive fields (Brooks, Rudomin & Slayman, 1961; Mountcastle, 1957). In the present study surround inhibition could be shown for adult rat forepaw units in S 1, and what appears to be the virtual replacement of these areas by surround excitation at 7 days of age. It is implied that a deficit of surround inhibition at an early age may be responsible for large receptive fields. An investigation of the ontogenesis of inhibitory mechanisms in the lemniscal systems would be useful in this respect. The responses reported here probably were conveyed by the lemniscal system since they were virtually all of the 'fixed field' type. The latter are claimed to be typical for cells activated by the lemniscal system (Brooks et al. 1961). 534

Latency of responses The latency of responses of SI cortical cells to natural cutaneous stimulation was considerably longer at 7 days of age when compared with adults, Similar observations have been noted by Rubel (1971) and Persson (1973) for immature S 1 cortex in the cat and sheep respectively. Since Rubel used electrical stimulation of the skin it seems improbable that these long latencies could be attributed to immature receptor function. Jacobson (1963) and Davison & Dobbing (1966) have shown that myelination is poor in the neonate rat and evidently this must be a contributory factor. At present there is little evidence on synaptic delays in immature ascending systems although it has been suggested they are abnormally long (Purpura et al. 1965). Inordinately long routes for activation of S 1 cortex in immature rats seems possible since the temporal dispersion of

535 SOMATOSENSORY CORTEX OF NEONATAL RAT activity in single units following peripheral stimulation often exceeded 500 msec in the present investigation. After-bursts of activity also were fairly common. Influence of stimulus repetition rate Inordinately long intervals between stimuli were required to obtain consistent activation of S 1 cortical cells at the 7 day stage. Most cutaneous areas could not be stimulated more than once per 15 sec without a decrease in response probability. In contrast to this it was found that spontaneous activity in SI cells at this age recovered to prestimulated levels within 2-4 sec after single cutaneous stimuli. Elements affected during prolonged decrease in transmission ability after tactile stimulation are not, therefore, the same as those causing spontaneous activity. This finding is in contrast to the suggestion (for immature visual cortex) that long interstimulus intervals are necessary due to 'fatigue' of immature cortical cells themselves (Hubel & Wiesel, 1963). Otherwise the excitatory synapses for spontaneous and evoked activity must presumably be qualitatively different. Work in progress in this laboratory indicates that the central portion of the gracile nucleus can faithfully follow stimulus repetition rates of 3/sec at 7 days of age. Thus it seems probable that the answer to 'fatigue' in the immature system will be found in the thalamus. Wherever the answer lies the somatosensory cortex of the immature rat apparently does not possess machinery for adequate interpretation of cutaneous stimuli at frequencies higher than about 5 or 6/min.

Spontaneous activity Although no previous investigations have been carried out on unitary activity in immature rat cortex, a paucity of spontaneous activity in immature mammalian cortex has been previously noted (Hubel & Wiesel, 1963; Hyvairinen, 1966; Krnjevid, Randic & Straughan, 1964; Huttenlocher, 1967; Huttenlocher & iRawson, 1968; Armstrong-James, 1973; Rubel, 1971; Persson, 1973). This is unlikely to be attributable to anaesthesia, since in the study on kittens by Huttenlocher & Rawson (1968) and that by Persson (1973) on foetal sheep, no anaesthetic was used and spontaneous activity was also infrequent. Difficulties in evoking activity in immature cortex have been encountered by all workers in the field. For example, Hubel & Wiesel (1963) describe 1 week old kitten striate cells as ' grudgingly responsive'; a similar problem was found in the present investigation. One possibility is that only a small proportion of cortical cells are adequately synaptically activated to cause them to discharge, either spontaneously, or in response to cutaneous stimulation. Synaptic development of rat cortex is poor at 7 days of age (Johnson & Armstrong-James, 1970)

M. ARMSTRONG-JAMES 536 and transmitter stores available for activation of cortical cells are only at about 4 % of the adult level (Armstrong-James & Johnson, 1970). The present study showed a clear difference between the types of spontaneous activity in adult and immature S 1 cortical cells as judged by the interspike interval histograms produced at the two ages. The great majority of spontaneously active adult cells produced long-tailed skew i.h.s classically associated with significant inhibitory mechanisms operating on the cell (Stein 1965). I.h.s produced by the spontaneously active immature cells were conversely approximately normal showing a lack of long intervals. Cutaneous stimulation caused the appearance of long interspike intervals in the immature cells which were associated with periods of inhibition of spontaneous activity. These observations strongly imply that inhibitory mechanisms contribute more to evoked activity in immature cortex than to spontaneous activity. There is, however, no evidence as to whether these mechanisms are of intra- or extracortical origin. Andersen and his co-workers have shown that cyclic activity in mature somaesthetic cortex, involving successive phases of excitation and inhibition, can depend on 'pace-maker activity' generated in lateral thalamic nuclei, included somaethetic thalamus (Andersen & Sears, 1964; Andersen, Andersson & L0mo, 1967; Andersen & Andersson, 1968). The type of cyclic activity they have described was seen in the present investigation in S 1 cortical cells in response to cutaneous stimulation at both ages, but was much more common in immature cells. It was somewhat surprising that no sign of cyclic activity was seen in the spontaneous activity of immature cells until they were activated by cutaneous stimulation. Indirect evidence of inhibitory mechanisms contributing to evoked cyclic activity in the immature cells is provided by the finding that the first phase of 'inhibition' following cutaneous stimulation was to a mean of 65 + 11-4 % of the spontaneous activity of eight cells investigated. Later sponperiodic decreases in taneous firing were nearly always apparent in the p.s.t.h.s produced by those cells (Fig. 22B, D, E and F). In conclusion, the results suggest that inhibitory mechanisms probably either within S 1 cortex or thalamus are brought into play on spontaneously active 7 day old S 1 cortical cells, in response to stimulation of cutaneous receptors. At this age these mechanisms appear to contribute more to cyclic activity than they do at maturity. I am indebted to T. G. Barnett for valuable assistance in design and construction of apparatus. I should also like to thank Manu Patel for his energetic and able assistance in some of the experiments. I am grateful to Professor K. W. Cross for his advice on the script and enabling me to draw on the resources of his department at all times.

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REFERENCES

ANDERSEN, P. & ANDERSSON, S. A. (1968). Physiological Ba8is of the Alpha Rhythm. New York: Appleton, Century and Crofts. ANDERSEN, P., ANDERSSON, S. A. & L0MO, T. (1967). Some factors involved in the thalamic control of spontaneous barbiturate spindles. J. Physiol. 192, 257-281. ANDERSEN, P. & SEARS, T. A. (1964). The role of phasing of spontaneous thalamocortical discharge. J. Physiol. 173, 459-4. ANGEL, A. (1967). Cortical responses to paired stimuli applied peripherally and at sites along the somato-sensory pathway. J. Physiol. 191, 427-448. ARMSTRONG-JAMES, M. A. (1965). Histological and neurophysiological studies on the postnatal development of the rat's cerebral cortex. Ph.D. Thesis, University of Bristol. ARMSTRONG-JAMES, M. A. (1973). Foetal and Neonatal Physiology. Proceedings of the Sir Joseph Barcroft Centenary Symposium, ed. ComLINE, R. S., CROSS, K. W., DAWES, G. S., NATHANIELSZ, P. W., pp. 28-32. Cambridge: University Press. ARMSTRONG-JAMES, M. A. & JOHNSON, R. (1970). Quantitative studies of postnatal changes in synapses in rat superficial motor cerebral cortex. Z. Zellforsch. microsk. Anat. 110, 559-568. BARLOW, H. B. & PETTIGREW, J. D. (1971). Lack of specificity of neurones in the visual cortex of young kittens. J. Physiol. 218, 98-100P. BOLLES LEE, A. (1950). The Microtomist's Vade-mecum, 2nd edn., ed. GATENBY, J. B. & BEAMS, H. W. Philadelphia: Blakiston. BROOKS, V. B., RuDOMIN, P. & SLAYMAN, C. L. (1961). Peripheral receptive fields of neurons in the cat's cerebral cortex. J. Neurophysiol. 24, 302-325. CALEY, D. W. & MAXWELL, D. S. (1968). An electron microscopic study of neurons during postnatal development of the rat cerebral cortex. J. comp. Neurol. 133, 17-44. CARTER, M. C., HOLMES, 0. & HoucHIN, J. (1969). Spatial distribution of electrical potentials evoked in the cerebral cortex of rats by peripheral stimulation. J. Physiol. 201, 16P. DAVISON, A. N. & DOBBING, J. (1966). Myelination as a vulnerable period in brain development. Br. med. Bull. 22, 40-44. ELLINGSTON, R. J. & WILCOTT, R. C. (1960). Development of evoked responses in visual and auditory cortices of kitten. J. Neurophysiol. 23, 363-375. GROSSMAN, C. (1955). Electroontogenesis of cerebral activity. Archs Neurol. Psychiat., Chicago 74, 186-202. HUBEL, D. H. & WIESEL, T. N. (1963). Receptive fields of cells in striate cortex of very young visually inexperienced kittens. J. Neurophysiol. 26, 994-1002. HUTTENLOCHER, P. R. (1967). Development of cortical neuronal activity in neonatal cat. Expl Neurol. 17, 247-262. HUTTENLOCHER, P. R. & RAWSON, M. D. (1968). Neuronal activity and AT Pase in immature cerebral cortex. Expl Neurol. 22, 118-129. HYvXRINEN, J. (1966). Analysis of spontaneous spike potential activity in developing rabbit diencephalon. Acta physiol. scand. suppl. 278. JACOBSON, S. (1963). Sequence of myelination in the brain of the albino rat. A. Cerebral cortex and related structures. J. comp. Neurol. 121, 5-29. JOHNSON, F. R. & ARMSTRONG-JAMES, M. A. (1970). Morphology of superficial postnatal cerebral cortex with special reference to synapses. Z. Zellforsch. milkrosk. Anat. 110, 540-558. KRNJEVI6, K., RANDI6, M. & STRAUGHAN, D. W. (1964). Unit responses and inhibition in the developing cortex. J. Physiol. 175, 21 P.

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M. ARMSTRONG-JAMES

MARTY, R. (1962). Developpement post-natal des responses sensorielles du cortex cerebral chez le chat et le lapin. Aspects physiologiques et histologiques. Arch8 Anat. micro8c. Morph. exp. 51, 129-264. MOUNTCASTLE, V. B. (1957). Modality and topographical properties of single neurons of cat's somatic sensory cortex. J. Neurophy8iol. 20, 408-434. MOUNTCASTLE, V. B., DAvIEs, P. W. & BERMAN, A. L. (1957). Response properties of neurons of cat's somatic sensory cortex to peripheral stimuli. J. Neurophy8iol. 20, 374-407. PERSSON, H. E. (1973). Foetal and Neonatal Phy8iology. Proceedings of the Sir Jo8eph Barcroft Centenary Sympo8ium, ed. COMLINE, R. S., CROSS, K. W., DAWES, G. S. & NATHANIELSZ, P. W., pp. 20-27. Cambridge: University Press. PURPURA, D. P., SHOFER, R. J. & SCARFF, T. (1965). Properties of synaptic activities and spike potentials of neurons in immature neocortex. J. Neurophy8iol. 28, 925-942. RUBEL, E. W. (1971). A comparison of somatotopic organisation in sensory neocortex of newborn kittens and adult cats. J. comp. Neurol. 143, 447-480. SCHERRER, J. & OEcoNoMos, D. (1954). Responses corticales somesthesiques du mammifere nouveau-n6 compares a celles de l'animal adulte. Etud. neo natal. 3, 199-216. SHOLL, D. A. (1953). Dendritic organization in the neurones of the visual and motor cortices of the cat. J. Anat. 87, 387-406. STEIN, R. B. (1965). A theoretical analysis of neuronal variability. Biophy8. J. 5, 175-194. THAIRU, B. K. (1971). Post-natal changes in the somaesthetic evoked potentials in the albino rat. Nature, New Biol. 231, 30-31. WELKER, C. (197 1). Microelectrode delineation of fine grain somatotopic organization of SmI cerebral neocortex in albino rat. Brain Res. 26, 259-275. WIESEL, D. H. & HUBEL, T. N. (1963). Single cell responses in striate cortex of very young visually inexperienced kittens. J. Neurophy8iol. 26, 994-1002. WOOLSEY, C. N. & LE MEssuIRIER, D. H. (1948). The patterns of cutaneous representation in the rat's cerebral cortex. Fedn Proc. 7, 137-138.