Postnatal development of identified medium-sized caudate spiny neurons in the cat

Developmental Brain Research, 24 (1986) 47-62 47 Elsevier

BRD 50308

Postnatal Development of Identified Medium-Sized Caudate Spiny Neurons in the Cat

M.S. LEVINE, R.S, FISHER, C.D. HULL and N.A. BUCHWALD

Mental Retardation Research Center and Brain Research Institute, University of California at Los Angeles, Los Angeles, CA 90024 (U.S.A.)

(Accepted June 1 lth, 1985)

Key words: neostriatum - - development - - electrophysiology - - horseradish peroxidase - - spiny neuron - - Golgi impregnation - - local axonal collateral

The morphology of intracellularly recorded neurons in the cat caudate nucleus (Cd) was studied during postnatal development. Af- ter intracellular recording of evoked responses in these neurons, horseradish peroxidase (HRP) was injected iontophoretically through the recording micropipette, Fifty-eight Cd neurons in cats ranging from 6 days of age through adulthood were identified mor- phologically. All of the recovered Cd cells were medium-sized spiny neurons. The basic somatodendritic morphology of these neurons was evident in the youngest kittens. The most striking morphological change was the postnatal formation of an extensive local axonal collateral plexus. The development of these local axonal collaterals was also quantified with computer assistance in medium-sized Cd spiny neurons selected from silver-impregnated material. This analysis showed that the major development of the branches of this local plexus occurred between birth and 3-4 months of postnatal age. Data from both the HRP-filled and silver-stained axons indicated that the postnatal growth of the local axonal collaterals of the medium spiny cells was associated with the elaboration and increasing prevalence of evoked inhibitory postsynaptic potentials in Cd neurons.

INTRODUCTION

Our laboratory has investigated the neurophysiological and anatomical characteristics of neurons in the developing and mature caudate nucleus (Cd) of the cat. In the adult, Cd neurons respond to activation of their monosynaptic inputs with an initial excitatory postsynaptic potential (EPSp)6as,16. This exci- tation is typically followed by an inhibitory postsynaptic potential (IPSP). In the referenced papers, we hypothesized that the inhibitory potential results from local inhibitory interactions among neighboring Cd neurons. In developmental neurophysioiogical studies, we showed that Cd neurons respond to activation of their major inputs at birth24,25,28, 29. With maturation proportionately more neurons respond, response latency decreases, synaptic security increases and the type of evoked response changes markedly. Intracellular recordings indicated that during the first few postnatal weeks, most responses consist of simple excitations. During the first two

postnatal months, the inhibitory postsynaptic potentials that follow these excitations in adult animals mature slowly29. Thus, the characteristic excitatory-inhibitory postsynaptic potential sequence (EPSP- IPSP) recorded intracellularly in adult Cd neurons when their monosynaptic inputs are activated is not well established until late in the postnatal development of the kitten.

In a parallel series of morphological studiesl,13a8 we showed that extensive growth of dendritic branches of Cd spiny neurons occurred between birth and 5 months. Length of dendritic segments, radius of the dendritic field and the density of spines on distal dendritic branches increase markedly during this period. In studies using transport methods, we demonstrated that both afferent and efferent connections of the Cd were present and functional in newborn cats 5,7.t0-13.

In our previous research on postnatal development of the Cd, physiological and morphological methods were used independently. The current experiment

Correspondence: M.S. Levine, Mental Retardation Research Center, 760 Westwood Plaza, Room 58-258, University of California at Los Angeles, Los Angeles, CA 90024, U.S.A.

0165-3806/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

48

was designed to assess the morphological features

and the characteristic electrophysiological responses

to afferent activation in single Cd neurons at differ-

ent developmental ages. Horseradish peroxidase

(HRP) was injected into impaled neurons after

evoked responses had been assessed by intracellular

recordings. This neuronal marking technique was

first used in the basal ganglia by Kitai et al. to delin-

eate the somatodendritic and axonal morphology of intracellularly recorded Cd neurons21, 22. The results

of our analysis of identified Cd neurons suggest that

the local axonal collateral plexus of the medium-

sized Cd spiny neuron is immature at the same early

postnatal periods in which the inhibitory component

of evoked responses in these cells is also immature.

In order to provide more information on the devel-

opment of the local axonal plexus, we also examined

and used computer assistance to quantify the extent

of local axon collaterals in silver-impregnated medi-

um-sized spiny Cd neurons.

MATERIALS AND METHODS

Electrophysiology and HRP morphology Animals. Twelve kittens and 9 adult cats were used

in this study. These animals were divided into 4 age

groups: 1-10 days (3 kittens), 11-20 days (5 kittens),

21-50 days (4 kittens) and adults (9 cats, >1 year of

age). The day of birth was considered as the first day

of age. Kittens were obtained from the UCLA Men-

tal Retardation Research Center cat breeding col-

ony. Surgery. Surgical procedures for acute electro-

physiological kitten and adult cat preparations have

been detailed previously 6,29. All animals were anes-

thetized during the recording experiments by the

continuous use of respiratory mixtures of halothane

and N 2 0 - O 2. To permit a direct approach to recording sites in the Cd, the overlying neocortex and cor-

pus callosum were aspirated. After the placement of each recording electrode into the exposed Cd, the cavity was capped with low-melting-point paraffin in

order to stabilize the well and the electrode mechani-

cally. Stimulating and recording. Stimulating electrodes

were inserted at 3 sites ipsilateral to the recorded Cd: (1) precruciate gyrus (Cx), (2) central medial and

parafascicular nuclei of the thalamus (Th) and (3)

substantia nigra (SN).

Recording electrodes (glass micropipettes filled with a solution of 0.5 M KC1 to which 3-5% w/v Sig-

ma Type VI HRP dissolved in (L5 M Tris-HCI buf-

fered at pH = 7.6 at 36 °C was added ~ were bevelled at the tip to an angle of 30 ° (50-2(10 Mr2 resistance in the brain I. Postsynaptic potentials evoked in Cd neu-

rons by electrical activation of C×. Th and SN were

amplified, monitored on an oscilloscope and record-

ed on FM tape for subsequent analx sis The most fre-

quently occurring responses of Cd neurons to activa-

tion of their monosynaptic inputs were depolariza-

tions or depolarizations followed b~ hyperpolallza-

tions. For the present experiments, the depolariza-

tions evoked bv the stimuli were considered to be

EPSPs while the hyperpolarization~ were considered to be IPSPs.

HRP injecnons were produced bv applying 5 - t5

nA positive DC pulses of 100 ms duration at 5 Hz.

Neurons were filled by injections that ranged between 5 and 50 nA/min A maxnnum of 3 neurons

were impaled and marked in each Cd of each animal.

Histology. After the final in iection of HRP into a

neuron, the animal was allowed to survive for at least 1 11 prior to sacrifice. Stimulating electrode tip sites

were marked by small electrolyuc lesions for subse-

quent histological determination. Animals were then

treated with heparin, killed with an overdose of sodi-

um pentobarbital and perfused transcardialty with NaC1, paraformaldehyde-glutaraldehyde fixative

and sucrose. Serial frozen sections (100 urn) were cut in the cor-

onal plane through the Cd and the stimulating sites.

Marked neurons were recovered in both kittens and

adults bv different histochemical methods for the de-

tection of peroxidase activity:~. Initially, we em-

ployed 3,3',5,5'-tetramethylbenzidine /TMB) as the chromagen in the HRP assay m order to maximtze

the sensitivity of the procedure and yield as many recovered neurons as possible 2~. However. the TMB reaction product was unstable, particularly in very young animals {< 30 days of age) where it tended to fade within 24-48 h after processing. Because of this

tendency, ontv a few neurons at the beginning of these experiments were stained with TMB. Benzi- dine dihydrochloride (BDHCI ~ or 3.5-diaminoben- zidine (DAB: coupled with glucose oxidase peroxide generation I GODI and cobalt enhancement ~) were

49

utilized as chromagens for most of the neurons ana-

lyzed in this investigation. All of these HRP assays

revealed similar axonal morphology in the marked Cd neurons, but the DAB method produced the best

resolution of somatodendritic morphology in all age

groups.

Silver impregnation and computer-assisted quantifica-

tion of axons of Cd neurons Animals and histological procedures. The age

groups used were 1-3 (3 kittens) and 114-124 days

(2 kittens). After phenobarbital overdose, animals

were sacrificed by vascular perfusion with 10% neu- tral buffered formalin. Tissue blocks were dissected

from the Cd and processed by standard rapid Golgi methods 18. Only well-impregnated tissue that satis-

fied all the criteria for quantitative analyses was in-

cluded in this study ~0. Neuron selection. A total of 30 Cd neurons were

analyzed; 10 cells at 1-3 days and 20 cells at 114-124

days. All cells were classified as medium-sized spiny

neurons on the bases of dendritic spines and somatic

diameters smaller than 25 ,urn. In the youngest animals, soma size was the major determinant of cell se-

lection since dendritic spines were often sparse. All

selected neurons had a main axon that could be fol-

lowed for up to 150/am and at least two major collate-

rals emanating from the main stem axon that re-

mained in the section. Selected neurons were located

in the head of the Cd in the same regions that yielded

the HRP identified cells. Computer methods. The soma, initial dendritic

segments and the extent of the local axonal plexus

were drawn at 450 x total magnification. The draw-

ing was transferred to an electromagnetic tablet

(Apple Graphics Tablet) which was interfaced with

an Apple 2e computer. The branches of the axonal plexus were traced with an electromagnetic pen. All

measurements were made in two dimensions. At the

TABLE I

Summary of recovered neurons

magnification employed for these drawings the preci-

sion of this system was about 1/~m. Thus, movement

of the pen across one tablet unit was equal to 1 ~m.

The length of each axonal branch, the number of ma-

jor collaterals emanating from the main axon, the

number of branches in each major collateral, the to-

tal collateral length, the maximum radius of each ma-

jor collateral from the center of the soma and from its

point of origin on the main axon and the length of the

main axon were measured. These values were deter-

mined for each cell and then were averaged for all cells in each age group.

RESULTS

Electrophysiology and H R P morphology

!ntracellular recordings of evoked responses to af-

ferent activation and subsequent morphological

identification were obtained from 58 recovered neu-

rons located in the head of the Cd of kittens and adult

cats. All these cells had the anatomical characteris-

tics of medium-sized spiny neurons. Spines were al-

ways evident, particularly on the more distal dendrit-

ic branches. Cell recovery. HRP-filled neurons were recovered

in 12 kittens of 6-50 days of age and 9 adult cats

(Table I). Data from kittens and cats were divided

into 4 age groups: 1-10 days, 11-20 days, 21-50

days and adults. These age groups were based on our

previous report 29 showing that the evoked IPSP of

the EPSP-IPSP sequence recorded in kitten Cd neu-

rons develops from 21-60 days of age.

In the 12 kittens (Table I), 48 neurons responding

to activation of one or more of the tested Cd inputs

received intracellular injections of HRP. Of these

cells, 27 (56%) were identified. In 4 cases, the den-

dritic processes were present but the somata had dis-

integrated or been 'blown apart' by the injection. In 5

cases, 2-4 spiny neurons in close aggregation were

Age Ca~ N e u r o n s N e u r o n s Neurons Blown Multip& Single %Re- %Measures recorded injec~d recovered somata injections n e u r o n s covered taken

1-10 days 3 13 13 3 0 0 3 23 23 11-20 days 5 16 16 12 2 3 7 75 44 21-50 days 4 22 19 12 2 2 8 63 42 Adult 9 51 50 31 6 8 17 62 34

g 10 Day

50

f.

~ ~uy

I,,1 13ay

100 ~m

Fig. 1. A developmental series of the somatodendritic morphology of medium-sized spiny Cd neurons. All drawings are camera lucida reconstructions obtained from serial sections of identified neurons. The projections of the reconstructed neurons are in the coronal plane. Two medium-sized spiny neurons of kittens of 10 days of age are shown in a and b. The overall somatodendritic appearance of neuron a was more mature than neuron b in that the dendritic field was fuller, dendritic processes were longer and dendritic spines were slightly more prevalent. Neuron c is a medium-sized spiny cell obtained from a 13-day-old kitten. It has a polarized dendritic field and bipolar somatic shape. By 20 days (neuron d}. the appearance of the dendritic field is more adult-like. Neuron e taken from a 30- day-old kitten, shows a nearlv mature dendritic field appearance. Neuron f is a typical medium-sized spiny Cd cell from an adult cat. The same scale applies to all reconstructions

s ta ined . T h e r e m a i n i n g 18 cases c o n s i s t e d of s ingle

iden t i f i ab le sp iny n e u r o n s in wh ich the s o m a t a a n d

dend r i t i c f ie lds we re w e l l - d e l i n e a t e d I see T a b l e I for

c a t e g o r i z a t i o n by age) . M o r p h o l o g i c a l m e a s u r e -

m e n t s we re o b t a i n e d f r o m this g r o u p In t he k i t t ens ,

67c~ (12/18) of t he i den t i f i ed n e u r o n s h a d s t a i n e d ax-

ons.

In adu l t cats (-Fable I) , 50 n e u r o n s r ece ived in t ra -

ce l lu lar i n j ec t i ons of H R P . T h i r t y - o n e ( 6 2 % ) were

r e c o v e r e d . O f t h e s e . 6 h a d "b lown ' s o m a t a , 8 consis-

t ed of mu l t i p l e sp iny n e u r o n s ( 2 - 5 cells} a n d 17 con-

s is ted of s ingle i den t i f i ab l e sp iny n e u r o n s in which the

s o m a a n d d e n d r i t i c f ie ld w e r e w e l l - d e l i n e a t e d . T h e

s ame p r o p o r t i o n of C d n e u r o n s was r e c o v e r e d m

adu l t s a n d k i t t en s of 1 1 - 5 0 days of age. In adu l t cats .

7 6 % (13/17} of t he n e u r o n s h a d s t a i n e d axons .

Morphological features of recovered neurons. The

m o r p h o l o g i c a l f e a t u r e s of i den t i f i ed sp iny Cd neu -

rons in k i t t en s a n d adu l t cats a re i l l u s t r a t ed in Figs. l

( s o m a t a a n d d e n d r i t e s ) a n d 2 ( local a x o n a l plexus}.

Fig. 3 shows e l e c t r o p h y s i o l o g i c a l r e c o r d s of e v o k e d

r e s p o n s e s in 3 o f t h e s e C d n e u r o n s ,

Somata and dendrites. All of t he H R P iden t i f i ed

cells we re m e d i u m - s i z e d sp iny ( ' d n e u r o n s . T h e re-

f.

51

10 Day 20 Day

L ~. \ \ ~ 30 Day

Adult 100 ~u.m

Fig. 2. A developmental series of the intrastriatal axonal morphology of medium-sized spiny Cd neurons. All drawings are camera lucida reconstructions obtained from serial sections of identified neurons, The projections of the reconstructed neurons are in the coronal plane. The letters beside each axonal reconstruction correspond to the somatodendritic reconstructions of the same cells shown in Fig. 1. In each axonal reconstruction, the somata and primary dendrites are displayed in order to delineate the origin of the stem axon. The same scale applies to all reconstructions. In neuron a (10-day kitten) the stem axon originates from a primary dendrite. A small collateral with a bulbous ending branches about 50#m from its site of origin. In neuron d (20-day-old kitten) the stem axon arises from the soma and gives rise to two short collaterals. In neuron e (30-day kitten) numerous intrastriatal collaterals emanate from the stem axon, branch repeatedly and form an elaborate local collateral plexus. Axonal varicosities were evident along the length and ends of the local collaterals. In neuron f (adult) the local intrastriatal plexus is illustrated. The stem axon is thick and runs distally after looping on itself. Numerous proximal collaterals arise to form an axonal plexus that is roughly contiguous with the dendritic field of the parent cell body. More distally, a single collateral arises.

covered neuronal cell bodies had spherical or oblate

shapes (Figs. 1 and 4). The somatic areas of the re-

covered Cd neurons ranged between 100 and 160

#m 2 and increased by 21% between 1-10 days of age

and adulthood (Table II). In all groups, the majority

of the recovered cell bodies of Cd neurons were

spine-free. However, a small proportion (about

20%) of the neurons identified in each age group had

sparse somatic spines. These latter neurons also had

sparse spines along their primary dendrites.

There were no developmental changes in the num-

ber of dendritic processes (5-7 primary dendrites)

originating from the cell bodies of the recovered Cd

neurons (Table II). Each dendrite branched at least

once and dendrites with more than 10 branches were

found in all age groups. The branching patterns and

lengths of the dendrites for each neuron varied wide-

ly. The dendrites of the Cd neurons occupied a

roughly spherical or oblate field in most cases al-

though extended oblong bipolar dendritic fields were

evident in some of the younger cells. The maximum

radius of the dendritic field increased markedly with

age. The estimated volumes of the dendritic fields of

the recovered Cd neurons (maximum length x width

× depth) also increased with age (696% increase be-

tween 1-10 days and adults).

52

a

10 DAY

d

20 DAY

20 m V ~ "~'~':';--£-L ° "-" "-~" -':%'-~" "----~'--

STIM CX

ADULT

x-~ F II ., I " i

L

120 mV

200 m's

Fig. 3 Oscillographic tracings of evoked intracellular responses to afferent actwation m mo.rphologically identified medium-sized spiny Cd neurons. Three consecutive evoked responses are shown from a neuron in a kitten of 10 days of age (a~. ~ neuron in a kitten of 20 days of age (d) and a neuron in an adult cat (f). The letters above each set of responses correspond to neurons illustrated in Figs. 1 and 2. In a. SN activation (at arrows) evoked an EPSP accompanied by an action potential. No [PSP occurred subsequent to the EPSP. In d. SN stimulation produced an EPSP and action potential. Again, no IPSP was evident after the depolarizahon. In f. the typical adult evoked EPSP-IPSP response to afferent activation was obtained. The calibration to the left is for recordings in a and d while that to the right is for recordings in f.

While the size and shape of the dendritic spines

were quite variable, their most common form was a

distal bulb (1 -2 ~m in diameter) with a thin connec-

tive neck (2-3 ~m in length, <1 ,um in diameter).

Longer filiform processes were also evident on den-

drites in the youngest cells. The most distal dendritic

segments had the greatest density of spines. Spree

density on these segments increased markedly during

postnatal development.

Axons. Regardless of age, more than 50% of iden-

tified Cd neurons possessed axonal elements that

were labeled by the HRP reaction product (Table II).

In these neurons, a single thick-caliber ( 1 -2 ~m core

diameter) stem axon arose from the cell body (67% I

or from one of the proximal dendritic segments

(33%) (Figs. 2 and 5). The stem axons could be re-

constructed from serial sections for 60-2000 um

(Fig. 2). Although their paths were tortuous and con-

voluted, they invariably projected laterally toward or

into the internal capsule.

At all ages. these axons exhibited some form of lo-

cal collateral plexus originating from the stem axon.

If branches were present, they were bifurcations.

The frequency of axonal swellings along the length or

at the ends of the axonal segments appeared to in-

crease considerably with age. In kittens of less than

30 days of age, a highly variable number of the ends

of the collateral branches had large (3-5 t~m in

length) cone-shaped profiles at their ends. These

profiles probably corresponded to growth cones 3°-3'~.

None of the apparent growth cones or more mature

terminal elements appeared to contact the dendritic,

somatic or axonal processes of their parent neurons.

The estimated volumes of the local axonal fields of

identified Cd neurons (maximum length x width x

depth) increased with age. One of the principal

sources of this expansion was an increase in the num-

ber of collaterals branching from the stem axon

(Table II). The number of proximal collaterals had

nearly attained adult values by 21-5(I days. Since the

local axonal terminal fields almost doubled in volume

after 50 davs of age, branching in the more distal axo-

nal collaterals and/or linear growth of the collateral

segments must also have contr ibuted to the estab-

lishment of the local axonal fields.

The earliest-formed collaterats arose from the

stem axons around 50 ~tm from thei r origin. In older

kittens, collateral branches were established both

proximal and distal to this point. However, collate-

rals did not originate outside of the dendritic fields

until after 50 days of age. Adult Cd neurons typically

demonstrated one or two of these distal collaterals.

I I

53

Fig. 4. Photomicrographic details of somata (left panels A, C and E) and distal dendrites (right panels B, D and F). The neuron por- trayed in A and B is from a 13-day-old kitten (DAB-GOD peroxidase histochemistry). In A, the densely stained aspinous somata (star) gave rise to numerous aspinous proximal dendrites. In B, sparse spinous processes (arrow) were evident in a thin, tapering distal dendritic segment. While the spine heads are clearly apparent, the spinous necks are short and more difficult to resolve. In C and D, a neuron obtained from a 20-day-old kitten is shown (BDHC peroxidase histochemistry). Occasional spines were found along the soma (star) and spines were evident beyond secondary dendritic branch points (arrow, C). In D, spines (arrow) were also found in some- what greater prevalence on thin tapering distal dendritic segments. An adult Cd neurons is shown in E and F (DAB-GOD peroxidase bistochemistry). The soma (star) and proximal dendrites were aspiny (E). In contrast, spines (arrow) were quite prevalent along uni- form diameter distal dendritic segments (F). The calibration applies to all panels.

54

T A B L E II

Quantitative results from HR P-filled neurons

Experimental values are means + S.E.M.

Age

1-10 d. 3 123 _+ 4* 11-20 d. 7 121_+6" 21-50 d. 8 143 +_ 4 Adult 17 149 + 2

* Statistically different from adult value (t-test, P < 0.05).

n Somatic and dendritic parameters

Somatic" Dendrites Maximum Dendritic Distal den- area (~m 2) (n) radius den- field volume dritic spine

dritic field × 10~ 4 mm 3 density (j~m) (spines~

lO ~m)

5 141 _+35* 52 +~ 37" 3 + t* 2(67) 1.5:~_ 1.5 + 5-6 219 _+ 19" 153 +_ 26* 4 ± I* 5 (71) 2.4 ± i . 5 5-6 273 +_ 19 260 q_: 5(1" 6 _+_ I * 5 (63) h.() _+ 1 9 5-7 305 +_ 13 414 & 46 10 +_ 1 13 (76) 8.5 ± 13

Axonal parameters

Output axon ,Number prin- Axomd fieht filled (%) cipal local volume

collateraL~ × 10 ~4 mnr; filled

3 -+1 ~ 7 ± 4 ~

118 _+_ 36 ~ 204 + 27

Fig. 5. Photomicrographic details of stained axonal elements . Panel A shows the intrastriatal e lements of a spraY Cd neuron of a kitten of 30 days of age ( B D H C peroxidase histochemistry). The thicker s tem axon arose from the soma (left triangle), goes out of the plane of focus and then reenters the focal plane to the right (right triangle). Thinner local collaterals extend from the stem axon. These local collaterals had enpassage varicosities (arrows). In panel B, the spiny dendrite (star) and local collaterals of a ~plny Cd neuron of a kitten of 50 days of age are shown ( D A B - G O D peroxidase histochemistry). En passage terminals were again evident (ar rows) . In C~ the stem axon (triangle) and the origin of several intrastriatal proximal collaterals are demonst ra ted in an adult spiny Cd neuron ( B D H C peroxidase histochemistry). The s tem axon originated from a proximal dendrite and coursed into the internal:capsule, t t emit ted numerous thinner local collaterals. Generally, thickenings were evident at the branch points of collaterals from the: s tem axon (star and upper triangle). In D, the en passage terminal e lements (arrows) of the local collaterals of an adult spiny Cd neuron are s h o w n ( D A B - G O D peroxidase histochemistry). The calibration applies to all panels.

TABLE III

Electrophysiological results from HRP-identified neurons

Age n Resting poten- Action potential (mV)* tial amplitude

(mY)*

1-10d. 3 -28_+ 2 18 + 2 ll-20d. 11 -32+3 18+2 21-50 d. 10 -40 + 3 28 + 4 Adult 23 -42 _+ 2 41 + 1

Age Stimula- Number Number EPSP EPSP- tion tested respond- IPSP site ing

1-10 d.**CX 3 3 6 (100) 0(0) SN 3 3

11-20d. CX 11 8 16(94) 1 (6) SN 5 3 TH 6 6

21-50d. CX 10 10 SN 1 1 10 (63) 6 (37) TH 5 5

Adult CX 18 17 SN 12 11 8 (19) 34 (81) TH 15 14

* Mean + S.E.M. ** TH sites not tested in this group.

At all ages examined, the bulk of the local collaterals were confined within the dendritic fields of the parent neuron.

Electrophysiology. Neurophysiological data were obtained from 24 identified spiny neurons in 12 kittens and 23 identified spiny neurons in 9 adult cats (Table III). These included all the identified single neurons and several cases in which multiple spiny neurons were stained. When the cell was impaled clear resting potential shifts (range o f -20 to -60 mV) occurred in all cases. Action potentials were usually greater than 40 mV in amplitude (Figs. 3 and 6). (As we have shown previously in kittens under 20 days of age 29 it is not uncommon to impale Cd neurons with action potential amplitudes of 20 mV or less. Data from such neurons are included in the present analysis since the action potentials generated by these cells were unipolar and clear shifts in membrane potential

occurred when inputs were activated.) Overall, average resting membrane potential values changed from less than -30 mV in the youngest kittens to greater than -40 mV in adults. The average action potential amplitudes increased from about 20 mV in the youngest kittens to greater than 40 mV in adults.

All Cd neurons responded to stimulation of one or

55

more of their major monosynaptic afferents: Cx, Th,

and SN (Table III). The postsynaptic potential re-

sponses recorded from Cd neurons by such afferent activation changed markedly with age. In the youngest animals (1-10 days), all responses consisted of pure depolarizations. An action potential usually occurred at the peak of the depolarization (Fig. 3a, Fig. 6). In the present data, sequences of depolarizations followed by hyperpolarizations did not occur in

kittens of 1-10 days. In a previous report (which included a much larger sample of neurons), about 20% of the responses obtained from this age group had depolarization-hyperpolarization sequences 29. As older

kitten neurons were sampled, EPSP-IPSP sequences appeared and increased in frequency (6% in the

l l -20-day group and 37% in the 21-50-day group). Examples of these potentials are shown in Fig. 3d for

a neuron recorded in a 20-day kitten (EPSP) and in Fig. 6 for a neuron recorded in a 16-day kitten (EPSP-IPSP). It is interesting to note that the neuron in the 20-day kitten had an extremely immature local axonal plexus (Fig. 2d). In contrast, the neuron

in the 16-day kitten had a plexus with 3 local collaterals and about 20 axonal branches. The observations in the present experiment are consistent with our pre-

vious report that the hyperpolarization of the EPSP- IPSP sequence develops late postnatally in Cd neurons. When hyperpolarizations were present in kittens under 50 days of age, they were usually of short- er duration and smaller amplitude than those observed in adults. In adults about 80% of the evoked postsynaptic responses of Cd neurons were EPSP- IPSP sequences (Figs. 3f and 6). This proportion matches the results of earlier reports6, 2~,22,25,28,29.

Quantification o f silver-stained axons o f Cd neurons

Quantitative analysis of the development of the axonal plexus of medium-sized spiny Cd neurons showed that the major development of the branches of this plexus occurred between birth and 3-4 months of postnatal age (Fig. 7). The morphogenesis

of the local axonal plexus was characterized by marked increases in the total length of the major collaterals and in the prevalence of branching. For this

quantitative analysis (Table IV), the axon was divided into a main stem and major collaterals of this main stem. There was a 169% increase in the total length of all collaterals. This increase in length reflected the

56 9 Day

ealmm=lelu ~ I ~ , ~ A

16 Day

A

Adult

20 msec

i! Ili

tt It

50"~sec

20 mV

Fig. 6. Oscillographic tracings of evoked intracellular responses of identified medium-sized spiny Cd neurons in 9- and 16-day kittens and an adult cat. Left panel shows expanded time base. Right panel shows slower sweepspeed of the same response. In each case stimulation of the Cx (arrow) evoked a depolarization accompanied by action potentials. In the 16-day kitten and adult cat the depolarization was followed by a hyperpolarization. Although not shown in the reconstructions, the neuron in the 9-day kitten had an immature local axonal collateral plexus. The main stem was present and one principal collateral emanated from the plexus. No branches were stained. The neuron in the 16-day kitten had a local axonalcollateral plexus with three principal collaterals and about 20 branches. The neuron obtained from the adult cat had an extensive local collateral axonal plexus with more than 8 principal collaterals and over t50 branches Vertical calibration refers to all traces.

addition of new branches to already existing collate-

rals. The number of major collaterals emanat ing off

the main stem axon did not increase notably (4.6 to

5.71. However, the number of branches/collateral in-

creased by 271% over the age period examined. The

total increase in length of each collateral was 118%.

Average branch length did not increase.

In order to further assess the increase in complexi-

ty of the local axonal plexus, a branch-order analysis

was performed (Fig. 8). The first branch off the main

stem axon was assigned an order number of 1. Num-

bering continued sequentially to the distal axonal

branches, being raised by one beyond each branch

point. In kittens of 1-3 days of age, branch orders

maximized at 1 -2 and rarely were more than third or

fourth order branches encountered. There were sig-

nificant increases m the complexity of the axonal

plexus. In the l14-124-day kittens, branch order

maximized at 3 - 6 and significant numbers of higher-

order branches (7 th-9 th order) occurred.

Several additional measurements revealed differ-

ences in the morphogenesis of local collaterals be-

tween neurons obtained from HRP and silver-im-

pregnated material. The average length of the main

stem axons diminished reliably with age in the silver-

stained cells (Table IV). This may have been due to

myelin accretion in the older animals which blocked

the silver impregnat ion of axonal processes closer to

the point of axonat origin 27. tn contrast to the volu-

metric expansmn of the local collateral terminal field

100#m

1-3 Day 114-124 Day

57

Fig, 7. Camera lucida reconstructions of the local axonal plexus of medium-sized Cd spiny neurons obtained from silver-stained material in 1-3 (left) and 114-124 day (right) kittens. The main stem axon is filled in for its initial course. Cell bodies are shaded for clarity.

suggested by the H R P identif ied Cd neurons, the

maximum radii of the s i lver- impregnated axonal col-

laterals from ei ther their somatic center or their point

of origin along the stem axon showed only minor in-

creases (Table IV).

There are a number of factors which could system-

atically affect these quanti tat ive results. Most of

these would lead to an underes t imat ion of the extent

of the plexus (i.e. loss of axonai mater ial lying out-

side the plane of section, incomplete impregnat ion of

axonal elements) . Due to the practical difficulties in reconstructing axons across tissue sections in silver-

s tained material , the extent of the processes may be

best reflected by the longest collateral evident in a

58

"FABLE IV

Quantitative parameters of axon growth

Lenght measures in ~m; experimental values are means z S.E.M.

A~e 1-3 days it4-124 days n 11) 20 Total length (all

collaterals) 438 _+ 50 1181 +_ t49" Number of collaterals/

neuron 4.6 ___ 0.4 5.7 _+ 0.5 Average collateral length 103 +_ 18 225 _+ 32* Branches/collateral 2.8 _+ 0.6 10.4 _+_ 2.1 * Average branch length 40 _+ 2.9 39 _+ 4.8 Maximum radius from center

of soma 104 ± 1(I 11i +_ 9 Maximum radius from main

stem axon 54 _+ 5 65 _+ 6 Length of main stem axon 374 _+ 31 230 _+ 29*

• Differences between the groups are statistically significant (t-test. P < 0.05).

single sect ion. B ranch lengths did not change o v e r

the d e v e l o p m e n t a l pe r iod we s tudied. Thus , the axon

col la tera l wi th the m a x i m u m n u m b e r of b ranches for

each cell was e x a m i n e d (Table V). As o b s e r v e d in

da ta f rom the en t i re p lexus , the length of this co l la te -

tn

-1 o

o

10

BRANCH ORDER I I I I I |

H 1-3 days 0--o 114-124 days

8 / "o-. . o / x

/ S p

, 'o 6 O" ~\

"o o

2 ~ "'o. -o

1 1 3 5 7 9 >10

O r d e r Fig. 8. Branch order analysis of the local axonal plexus of iden- tiffed medium-sized Cd spiny neurons obtained from silver- stained material in 1-3 and 114-124 day kittens. Each point represents the average number of collateral branches/neuron at each order. Branch order > 10 is an overflow bin and contains all orders beyond 10. Differences between the distributions were assessed with X 2 analysis and are statistically significant (X 2 = 32, df = 10, P < 0.001),

T A B L E v

Analvsis qf largest collateral

Length measures m urn: experimental values are mean,~ ~_. SE.M.

Age 1 -3 da~ 14-124 days Number of branches 5.9 _+ l.h 27.9 + 5.6" Total length 228 + 4~; 541 c 72 ~ Maximum radius from

centerofsoma 135 ! 15 132 z 14 Maximum radius from

main stem axon t l l z I() lt~; = 10

* Differences between the groups are statisticalh, significant It-test. P < ( I)5~

ral and the n u m b e r o f branches inc reased m a r k e d l y

as a direcl funct ion of age ( 137q/( and 373% respec-

t ively). The re were also no age- re la ted changes in

the m a x i m u m radii of this col la tera l f rom e i the r the

somat ic cen te r o r f rom their po in t of or igin a long the

s tem axon.

D I S C U S S I O N

T h e m a j o r f inding of this s tudy is that the axonal

m o r p h o l o g y of med ium-s i zed spiny Cd neu rons

changes m a r k e d l y dur ing pos tna ta l d e v e l o p m e n t .

The analysis of neu rons s ta ined with H R P indica tes

that the local axonal p lexuses are not p r o m i n e n t unt i l

app rox ima te ly 30 days o f age. The /ate pos tna ta l

morphogenes i s of local col la tera ls involves out-

g rowth , b ranch ing and en passage t e rmina l fo rma-

t ion. T h e quan t i t a t ive analysis p rov ided by the silver-

i m p r e g n a t e d mate r ia l indicates that the ex tens ive

f o r m a t i o n of b ranches that occurs pos tna ta l ly leads

to increases in col la tera l length . A l t h o u g h the re is a

m a r k e d increase m the n u m b e r of b ranches per col-

la teral , the fo rma t ion of s o m e m a j o r col la tera ls ap-

pears to occur be fo re bir th. Cor re l a t i ons b e t w e e n the

morpho log ica l d e v e l o p m e n t and neurophys io log ica l

d e v e l o p m e n t of e v o k e d synapt ic responses indica te a

re la t ionship b e t w e e n the occur rence o f hyperpo la r i z -

ing m e m b r a n e potent ia l s in these iden t i f ied Cd neu-

rons and the e l abora t ion of the local axona l col la tera l

plexus.

Development o f axons o f Cd spiny neurons

In the adult cat Cd . th in intrastr iatal col la tera ls

o r ig ina te f rom the s tem p ro j ec t i on axons of the medi -

um-s ized spiny Cd neu rons z0,2t. These local co l la te -

rals were i m m a t u r e in k i t t ens u n d e r 10 days o f age

59

and their morphogenesis proceeded during postnatal

development. This morphogenesis included in-

creased branching, lengthening and the formation of en passage swellings that probably constitute axonal terminals 30.

While there is an immature appearance of the local collateral axonal plexus in newborn kittens, the main stem axon is always present. Even in the youngest HRP material examined (6-10 days) and in the youngest silver-stained material (1 day), this axon could be traced toward the internal capsule. Addi- tional anatomical and physiological studies from our

laboratory have demonstrated that the stem axons of the medium-sized spiny neostriatal neurons project to other nuclei and form terminal fields there by the day of birth in the cat 10,11.13. These projection neu-

rons are capable of supporting both ortho- and retro- grade axonal transport and exert a functional influ- ence on pallidal and nigral neurons from birth to adulthood~0.24.

Correlation of electrophysiology and morphology of Cd spiny neurons

The findings of the present experiment indicate that there is a correlation between the postnatal morphological development of Cd neurons and their functional capacity. There is general agreement that afferents to the Cd produce initially excitatory effects on Cd spiny neurons6,16,17,22, 32. The results of the

present study and those of previous physiological experiments which were based on a larger sample of neurons 29 demonstrated that, in kittens of 1-20 days

of age, stimulation of any major Cd afferent produced an initial depolarization accompanied by an action potential. The morphological mechanism un- derlying this depolarizing potential must be rea- sonably mature at birth in the cat. A simple possibili- ty is that a great majority of the afferent excitatory synapses are present and in place at birth and these are probably localized to the dendritic shafts and spines. Ultrastructural studies indicate that asym-

metrical synapses occur on both spines and dendritic shafts of Cd spiny neurons in newborn cats 1. During postnatal development extensive terminal addition, synaptogenesis, dendritic process outgrowth and dendritic spine formation occur. Thus, the prevalence of extrinsic inputs and the area of postsynaptic membrane available for their reception increases

markedly as a direct function of age 1.7,18. These mor-

phogenetic alterations contribute significantly to im-

proved coupling of the cells with their excitatory in- puts23,25,28,29.

In contrast, both the hyperpolarization and the local collateral axonal plexus appear to be immature during the first several postnatal weeks. With increasing age, local collaterals form, their number in-

creases and more terminals en passage develop on these collaterals. It is during this period that Cd spiny neurons begin to display more frequent, greater am-

plitude and longer-duration evoked hyperpolarizations. We have hypothesized previously that the

IPSP of the EPSP-IPSP sequence is due to local inhibitory interactions among neighboring neostriatal neurons 4,6,16,29. The absence of inhibition, the imma-

turity of the local collateral plexus in newborn cats

and their subsequent parallel maturation support this hypothesis.

An alternative hypothesis of the origin of the IPSP based on the effects of lesion studies in the rat has re- cently been proposed 41-44. These authors agree that

a short-duration component of the inhibitory response is generated internally. However, a longer

duration component is said to be caused by disfacilitation arising from inhibition of tonically active excitatory neostriatal inputs from the cerebral cortex or thalamus 35,36,41,42,44. The initial activation of Cd neu-

rons produces inhibition via basal ganglia output

pathways to thalamus and cortex and/or via direct activation of local inhibitory networks in the areas stim- ulated. The activation of these inhibitory networks

causes a cessation of excitatory impulses to the neostriatum which produces a passive hyperpolarization of the neuronal membrane potential.

At present, the evidence for the existence of such disfacilitation in the Cd of the cat is not compelling.

In adult cats, disruption of the afferent connections by extensive thalamic destruction fails to alter the properties of the inhibitory response evoked by activation of the remnant direct inputs to the Cd 37. Dis-

ruption of corticocaudate afferents, in contrast, re- duces the amplitude of the hyperpolarization45. To explain our current results by the disfacilitation hypothesis, Cd output pathways must be incapable of activating or inhibiting their target regions and/or local inhibitory circuits in cortex and thalamus must not be functional during the first postnatal month. Devel-

60

opmenta l e lectrophysiological findings are not sup-

port ive of ei ther al ternat ive. Globus pallidus, ento-

peduncular and SN neurons are capable of respond-

ing to activation of the caudate even in newborn kiT- tens 1°,24. Thus, Cd output pathways are capable of

functioning at this t ime. Thalamic neurons are also

capable of being act ivated by st imulat ion of the Cd or

giobus pail idus in newborn kittens 14,29. In addit ion,

thalamic neurons are capable of generat ing large-

ampli tude and long-durat ion IPSPs in kittens under

10 days of age TM. Finally, in the neocortex, inhibi tory

events appear deve lopmenta l ly before excitatory

events or are more potent than exci tatory events ear-

lier in deve lopment 17,33 and thus disfacili tation could

occur during early deve lopmenta l periods. In sum-

mary, it appears that the hypothesis of an intrastria-

tal origin of inhibit ion in the Cd is both more con-

gruent with the empir ical evidence and more parsi-

monious than the disfacili tation hypothesis.

Previous studies of the deve lopment of basal gan-

glia neurons in monkeys 2-3-~ and cats 1.9,1~,3t have

shown that neurons at marked ly different stages of

maturi ty are present at early postnata l ages. In the

material examined in the present study, there was

also considerable variat ion in any one cat in lhe ma-

turity of the appearance of spiny Cd neurons and m

the size and the extent of axonal plexus. However , m

our youngest mater ia l ( 1 -3 days), we never encoun-

tered an axonal plexus that was as extensive as the

most complex arbor izat ion observed in 1 14-124 day

kittens. In the older material , the opposi te occa-

sionally occurred. In a number of cells examined at

114-124 days, a sparse local col lateral plexus similar

to that seen in the newborn kitten was present . It is

possible that this variat ion in the extent of the local

plexus underl ies some of the variat ion we observed in

the occurrence of the IPSP of the E P S P - I P S P se-

quences. For example we showed two neurons from

animals close to the same age (16 and 20 davs) that

had two different e lectrophysiological responses.

The neuron from the younger cat displayed an E P S P -

IPSP sequence and had a local plexus with several

major collaterals and branches. The neuron from the

20-day-old animal displayed only an EPSP and had a very immature local axonal col lateral plexus. It is ap-

parent from the present results and from our pre-

vious repor t 29 that the IPSP develops over a relative-

ly tong postnatal per iod in the cat. Similarly, if the

maturat ion of the local plexus underl ies the devel-

opment of the IPSP. at any given age some neurons

should be expected to have an extensive plexus and other would not.

Methodological variables"

It is possible that the immatur i ty of the local axonal

plexus in young kittens is related to sore e of the meth-

odological variables encountered when using mtra-

cellular injections of marker substances. It is appar-

ent in our studies that HRP was t ranspor ted into the

axons since the main stem axon was s tained for some

distance in both kittens and adults. However . the

physical size of the H R P may have precluded its en-

try into the f ine-diameter processes of immature col-

laterals. Thus. the local collateral plexus of the medi-

um-sized spiny Cd neurons of young kittens rnav extst

but be undetectable with the single cell identif icat ion

method used in the present stud;r. We believe this ex-

planat ion is not likely. First , dendri t ic spines were al-

wavs evident in our material . The spine necks were

thin (<1 um in d iameter) , approximate ly the same

size or smaller than identif ied collaterals of the local

axonal plexus. Second. extensive. )1 not comple tc ,

filling of similar fine collaterals has been demon-

s trated developmenta l ly in neurons m h ippocampal

brain slices and cultures of spinal cord neurons using

procedures similar to those of the current experi-

ment 3(),3~. Third. labeling into both growth cones and

distal collateral e lements occurred m some cases.

Thus. it seems likelv that if more of these processes

were present , they too would have been labeled. Yhis

evidence does not explain the occasional failure to

obtain any axonal labeling in some of the identif ied

neurons in each age group. However . since stmilar

propor t ions of neurons in all age groups had non-la-

beled axons, this was not a developmenta l variable.

There are technical considerat ions that may have

ssstematically affected the data ob ta ined from the

silver-stained material . It is apparent that we could

never reconstruct the entire local axonal plexus. We

used only one section and did nor trace axons from

section to section. Our est imates of axonal plexus

length and complexity are obviousl~ underest imates .

However , given that the axons are matur ing and

more branches are forming it would be expected that

more material would be omit ted in the older animals.

Thus, we would be biasing our da ta a~ainst the ~reat-

61

er col lateral complexi ty in the o lder animals . It is pos-

sible, as indica ted above for the H R P analysis, that

the local plexus is p resen t in very young ki t tens bu t

remains uns ta ined by the Golgi t echnique . This is

also unl ikely. Most of the evidence suggests that the Golgi stain works best on embryon ic and early post-

natal tissue 27. Myel in fo rmat ion which occurs over

the first m o n t h tends to impede such impregna- tion27,34.

ACKNOWLEDGEMENTS

This work was suppor ted by U S P H S Gran t s H D

05958 and A G 01558.

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Postnatal development of identified medium-sized caudate spiny neurons in the cat

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