Opposing effects of striatonigral feedback pathways on midbrain dopamine cell activity

Brain Research, 333 ( 1985/271-284 Elsevier

271

BRE 1O713

Opposing Effects of Striatonigral Feedback Pathways on Midbrain Dopamine Cell Activity ANTHONY A. GRACE* and BENJAMIN S. BUNNEY Departments of Psychiatry and Pharmacolog). Yale University School o] Medicine. New ltaven. (.'7"06510 I U. S.,4. )

(Accepted August 10th. 1984) Key words: substantia nigra - - caudate nucleus - - electrophysiology - - intracellular recording - dopamine neurons - - GABA --zona reticulata - - striatonigral pathway,

The existence of a striatonigral GABAergic pathway has been well established both anatomically and biochemicallv. During intracellular recording from identified DA neurons in vivo, stimulation of the striatum (100 .uA. 50 ,us pulses) elicits an inhibitory postsynaptic potential (IPSP) and a rebound depolarization. The IPSP is a short latency ( 1.8-2.2 ms) conductance increase to chloride, since: (1) the reversal potential is near the chloride reversal potential reported for other cells {-68 mV): (2) intracellular chloride injection progressively reverses the IPSP into a depolarization with a similar time course: and {3) the response of DA cells to systemic injection of the chloride channel blocker, picrotoxin, also exhibits a similar reversal potential. In contrast, during cxtraccllular recording, stimulation of the striatum at low levels of intensity (e.g. 2[) uA at 10 Hz) increases the firing rate of DA cells. Stimulation of the striatum will, in addition, elicit IPSPs in a subclass of substantia nigra zona reticulata neurons at the same latency as the IPSPs triggered in DA cells. These IPSPs also reverse with intracellular chloride injection. However. their amplitude is larger and their duration longer than observed in DA cells, and thcre is no depolarizing rebound. The late component of the IPSP in the zona reticulata neurons corresponds temporally to the rebound depolarization seen in DA cells in response to striatal stimulation. In addition, when recorded extracellularly, striatal stimulation will inhibit the firing of this class of zona reticulata interneurons at the same stimulation parameters that will excite l)A cells. These data suggest that striatal cells may send branched fast-conducting GABAergic projections to zona reticulata cells and DA cells. Furthermore, low levels of striatal stimulation can excite DA cells by preferentially inhibiting interneurons in the zona reticulata which are more sensitive to the inhibitory effects of GABA than are DA neurons. INTRODLCTION

shown to m a k e direct m o n o s y n a p t i c c o n n e c t i o n s with identified D A n e u r o n s 8~, or r e t r o g r a d e l y labeled ni-

A l m o s t 9(Ic~, of the a f f e r e n t input of the substantia nigra

arises

from

cells

located

in

the

stria-

tum'~,5v.s'~.7-L A l t h o u g h it was o n c e b e l i e v e d that the striatonigral p r o j e c t i o n s a r o s e f r o m only 5 % of the striatal cells 3-~.5s, m o r e

recent

investigations have

c o n c l u d e d that 3 ( l - 5 0 % of the cells in the s t r i a t u m project to the substantia nigra 7.15.29.as. This striatoni-

grostriatal zona c o m p a c t a n e u r o n s 7~'. M o s t striatonigral afferents, h o w e v e r , synapse p r e f e r e n t i a l l y in the zona reticulata region 3.a~,57',7°,73. T o date, t h r e e p u t a t i v e n e u r o t r a n s m i t t e r s

have

b e e n linked to these pathways: (1) 7 - a m i n o b u t y r i c acid ( G A B A ) ; (2) s u b s t a n c e p3,,.5~.55: and (3) the opiate-like p e p t i d e , d y n o r p h i n s5. T h e striatonigral

gral p r o j e c t i o n has b e e n c o n f i r m e d using m a n y techniques: Cl) a n t e r o g r a d e >s2 and r e t r o g r a d e *.l-s,ax.75,~v

ized of the nigral afferents. This G A B A e r g i c projec-

tracing t e c h n i q u e s ; (2) a n t i d r o m i c activation of stria-

tion was first d e s c r i b e d bv Precht and Y o s h i d a ~'~

G A B A e r g i c p r o j e c t i o n has b e e n the best c h a r a c t e r -

tal cells from the nigra>,~3,;~; (3) tracing of the axon

based on the analysis of nigral field p o t e n t i a l s trig-

of a single striatal cell to the nigra f o l l o w i n g intrasomatic striatal cell H R P injectionl'~: and (4) d e g e n e r a -

g e r e d by stimulation of the s t r i a t u m and their blockade by picrotoxin. B i o c h e m i c a l studies h a v e since

tion studies, w h e r e striatonigral n e u r o n s h a v e b e e n

confirmed

this

observation.

Thus,

dramatic

de-

• Present address: Department of Physiology and Biophysics, New York University Medical Center. 5511First Ave.. Ncv, York. NY 101116, U.S.A. Correspondence: B. S. Bunney. l)cpartments of Psychiatry and Pharmacology, Yale University School of Medicine. New I taven. C"I 1)65l(J. U.S.A.. tl{~16-XO93;85;$03.30 © It~85 Elsevier Science Publishers B.V. (Biomedical Division)

272 creases in the levels of G A B A and glutamic acid decarboxylase (a GABA-synthesizing enzyme~ within the nigra will occur lk)llowing lesions ot the striatum or sectioning of the striatonigral projecliOl.132.','L52.St,,¢,41.,,l.7t). Both z o n a conlpact;:t D A n e u r o n s and non-l)A zona reticulata cells are known to receive a G A B A e r g i c input. However. the zona reticulata cells are more sensitive to inhibition bv G A B A than are D A cells a0.,~s. Furthermore, preferential inhibition of a class of zona reticulata intcrneurons (ZR neurons) by low doses of systemically administered (}ABA agonists will disinhibit I)A cells and thus increase I)A cell activitv*L It is unclear at present how the striatonigral pathways modulate I)A cell firing, and questions have arisen concerning the net effect of striatal activity on DA neurons. Thus, on the one hand, there is a well-characterized striatonigral G A B A e r g i c pathway which directly innervates I)A neurons. ()n the other hand. there is evidence that striatal excitement may increase DA neuron firing". In this paper, the control of I)A cell firing b~ this striatonigral G A B A e r g i c pathway was examined using intracellular and cxtracellular recording techniques. These data have been reported m part m a symposium~ L

trom 2.0 mm glass tubing containing a Icy, strands t,i fiberglass stJ and filled with 2 M Na('l c~mI~unmg 2', Pontamine Sky Blue to aid in histologicai ~erihcatiem of the recording sites ~z. l h c electrutk rmpedancc measured 6-111 M£2 at lO()(J tlx. At the end ot czwh recording, a 30,uA cathodal currcm ~a,, passed through the electrode for 5 rain in order Io mark the recording site with a 50 .urn spot of blue d,,c. lntracellular recording electrodes ~erc pulled from 1.0 mm diameter WPI ()mcgadot glass tubing using a modified Narishigc vertical electrode puller and filled with 3 M potassium acetate. Electrode impedances were generally around 30-4tl ,x,.l~2 measured at 1000 |--lz. Only data ~htaincd from qable penetrations were used. A penetration wa~, defined as stable when: (1) the cell to be studied ~as hyperpolarized and inactive, or firing spontaneously at a rate similar to that observed extraccllularly for these neurons45: (2) action potentials exhibited amplitudes of 55 mV or greater: (3) the cell had a resting potential of 55 mV or greater; (4) the cell demonstrated the ability to generate multiple spikes in response to a depolarizing pulse: and (5) a stable input resistance above 15 Mff2 was observed.

Striatal stimulation M A T E R I A I . S AND M E T H O D S

All intracellular and extracellular recording experiments were performed in vivo in male SpragueDawley albino rats. The methods used were the same as those described previously 4-~-45. Briefly, rats were either: (I) anesthetized with chloral hydrate (400 mg/kg i,p. ) with supplemental anesthetic delivered by a needle secured into a lateral tail vein; or (2) anesthetized with chloral hydrate, paralyzed with gallamine and artificially respired via tracheal cannulae. Rats were mounted in a stereotaxic apparatus and their temperature monitored by a rectal probe and maintained at 37 °C. Electrodes were lowered through a burr hole overlying the substantia nigra region (1950 ,urn anterior, 2200 .urn lateralea). All drugs were administered via the lateral tail vein needle. Surgery was carried out in strict accordance with the

Guiding Principles in the Care and the Use q[ Animals. as approved by the Council of the American Ph.vsiological Society. I--xtracellular recording electrodes were pulled

Striatal stimulation was carried out in order to assess its effects on nigral neuron firing rates and membrane properties. For these studies, commercially available concentric stimulating electrodes were used (Kopf. model SNE-100). The coordinates used were 7600 um anterior and 3500.urn lateral ~'4, and 4500.um ventral to the brain surface. Stimulations were given in two ranges of intensity: 1 0 - 5 0 u A pulses delivered at 1-20 Hz and with durations of 5(1 us, or 10(I-200 u A pulses delivered at 20- 100 Hz and with durations of 50-200 .us.

Cell identification Dopamine cells were identified electrophysiologically by a number of criteria, including: ( 1) anatomical location; (2) firing pattern; (3) firing rate: (4) action potential waveform: (5) antidromic activation from the striatum; and (6) inhibition of firing by low doses of systemically administered DA agonists and reversal by D A antagonists ls.al.42-51. DA cells in the substantia nigra are located in the zona compacta region. just ventral to the medial lemniscus2".sL These

273 cells are characterized by a slow, irregular firing pattern (typically 2-6 Hz as) often intermixed with periods of burst firing, in which firing occurs in sequences of 3-10 spikes of decreasing amplitude and increasing duration 1~.42,46. DA cells can be antidromically activated from their terminal fields in the striatum at a conduction velocity of 0.5-0.6 m/s 4L42.51, a characteristic of their thin, unmyelinated axons. The action potential is irregular in shape, often consisting of a prominent positive-going initial segment (IS) spike followed by a biphasic (positive/negative) somatodendritic (SD) spike, with a total duration of 1.8-4 ms. A class of zona reticulata neurons (ZR neurons) which are hypothesized to be involved in the striatonigral feedback loop were distinguished during extracellular recording by the following criteria40: ( 1) location is typically within 101)um of the DA cells and ventral to the DA cell region: (2) they have a short duration (less than I ms) biphasic action potential which is often first observed as a monophasic negative spike: (3) these neurons respond to a footpinch with a short duration increase in firing rate: and (4) they respond to striatal stimulation or G A B A iontophoresis with a pronounced inhibition of spontaneous firing.

Reversal potential The reversal potentials '~ of drug-induced responses were determined in this and the next paper 47 in order to identify the ionic conductance changes underlying these responses. This was done by plotting the membrane voltage responses to 200 ms hyperpolarizing constant current pulses of various amplitudes. Regression lines were determined from these current-voltage plots before and after administration of the drugs in question. The point of intersection of the predrug and postdrug regression lines would thus occur at the membrane potential at which administration of the drugs would cause no change in membrane potential. This would be, by definition, the reversal potential of the ions involved in the response.

Histology At the conclusion of each extracellular recording experiment, Pontamine Sky Blue dye was iontophoresed from the recording barrel as described. The rat

was then perfused with isotonic saline followed by 10% formaldehyde in saline. The brain was removed, stored overnight in formalin solution, and cut into 50 um sections. These sections were stained with cresyl violet and neutral red, and examined microscopically to determine the location of the dye spot. RESULTS

Direct effects of striatal stimulation on nigral neurons recorded intracellularly The effects of stimulation of the striatum on DA neuron electrophysiology were studied using in vivo intracellular recording techniques. Stimulation with single pulses of current resulted in an inhibitory postsynaptic potential (1PSP) in DA neurons with a latency of 1.8-2.2 ms. The amplitude of the striatally elicited IPSP measured intracellularly in DA cells was 3.4 + 1.9 mV (n = 35, this and all following statistics are reported as mean + standard deviation) and typically exhibited rebound depolarizations. In an attempt to determine the ionic conductances involved in this response, the reversal potential of the IPSP was determined by observing the amplitude of the striatally evoked IPSP at different membrane potentials (Fig. 1). The membrane potential at which this hyperpolarization reversed to a depolarization was calculated to be -68.3 _+ 8 mV (n = 20). The calculated reversal potential for the striatallv evoked IPSPs was close to that reported for the chloride ion-mediated responses described in other preparations 5.25.,~. In order to determine if chloride was involved in this response, intracellular recordings were made from DA neurons with electrodes filled with potassium chloride (3 M). Increases in chloride conductances are normally inhibitory: thus, an increase in chloride conductance causes chloride to flow down its concentration gradient into the DA cell and results in a hyperpolarization. Using KCl-filled intracellular recording electrodes will cause chloride ions to diffuse from the electrode into the cell. This will reverse the concentration gradient of chloride ions across the membrane and invert the polarity of chloride-mediated responses 21. IPSPs elicited in DA cells impaled with KCl-filled electrodes consistently demonstrated a time-dependent decrease in amplitude followed by reversal into a depolarization (n = 8, Fig. 2A). Prolonged injection of chloride into

274

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+

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MEMBRANE POTENTIAL (mV)

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Fig. 1. Calculation of the reversal potential of the IPSP produced in DA neurons by stimulation of the striatum. A: stimulation ot the striatum (arrow, bottom group of traces) triggers a short latency IPSP along with the antidromically elicited IS spike. Intracellular injection of progressively higher levels of hyperpolarizing current (top) shifts the membrane potential of the DA neuron in the negative direction (bottom). This negative shift in membrane potential is accompanied by a decrease in the amplitude of the IPSP and finally a reversal in its direction to yield a depolarization. B: plot of the amplitude of the IPSP versus the membrane potential at which the IPSP was elicited to yield a regression line for this response. This IPSP reverses to a depolarization at -69.2 mV (where the regression line crosses the X-axis), which would be the reversal potential (or equilibrium potential) of the ions mediating the IPSP produced in DA cells by striatal stimulation.

275

A

!

10 mSeo

B

!

!

10 mSeo Fig. 2. Effects of intracellular chloride injection on the IPSP elicited in D A neurons by striatal stimulation. A: stimulation of the striaturn (arrow) during intracellular recording from a D A neuron elicits an IPSP in this D A neuron (bottom trace). W h e n an electrode containing KCI as an electrolyte is used, the diffusion of chloride into the D A cell progressively decreases the amplitude of the IPSP until it reverses direction. This is evidence that the IPSP elicited in D A neurons by striatal stimulation is mediated by a conductance increase to chloride ions. B: spontaneously occurring IPSPs are not typically observed during intracellular recording due to their long time course and low amplitude. However, with sufficient levels of intracellularly injected chloride ions, the IPSPs shift to larger amplitude depolarizing responses and may be observed occurring spontaneously, as shown here. T h u s , D A neurons appear to be under constant b o m b a r d m e n t with chloride-mediated IPSPs.

27~ I ) A cells led to the a p p e a r a n c e of numerous depolarizing potentials, which were assumed to be reversed spontaneously occurring IPSPs (Fig. 2B). since the~ occurred only with KCI-filled electrodes and could be reversed at m e m b r a n e potentials negative to zero mV (not shown). Picrotoxin is a specific blocker of chloride ion channels. A t t e m p t s were made to d e t e r m i n e the reversal potential of the picrotoxin-induced depolarization of D A cells to further test if chloride is indeed mediating this response. Due to the large i.v. injection volumes required, and muscle fasciculations and often convulsions associated with the use of this drug, only three cells yielded sufficient data to estimate the reversal potential of the p i c r o t o x i n - m e d i a t e d responses ( - 7 3 + 8 mV, Fig. 3).

Response oJ DA neurons to striatal stimulation observed during extracellular recording Despite the known direct G A B A e r g i c pathway to the nigra from the striatum and the IPSPs elicited in D A neurons from striatal stimulation, we have experienced difficulty in clearly d e m o n s t r a t i n g inhibition of the spontaneous activity of D A neurons by trains of striatal stimulation. At a relatively high current intensity (e.g. 500 ~ A or more), trains of stimulation delivered to the striatum will lead to some inhibition

PICROTOXlN

of D A cell firing H o w e v e l . it much to~ci ievci~ ~i stimulation arc used (average 20-50 uA. :,lJ u~ dttration al 2() Hz) there is an in( rea.se in the tirirlg talc (H D A neurons (27.7 + 14.9c4 incre,t,c )n ; a t e P < 0.01, n = 14). These lower current lcxcls arc i~ck)w the stimulation intensities required )t, antidromically activate D A neurons.

Responses o f non-DA nigral neurons to .~trtatal sttmulation During attempts to record from D A neurons intracellularly, a number of types of neurons in the zona reticulata ( Z R ) could be p e n e t r a t e d , thus allowing the comparison of their responses to striatal stimulation with those of D A neurons. Nearly all cells tested (confined mostly to those .just ventral m the zona compacta) responded to striatal stimulation with IPSPs of latencies identical to those found for striatally m e d i a t e d IPSPs ill I ) A neurons (approx. 1.5-2.5 ms). The amplitudes were consistently much larger than the amplitude of the IPSPs elicited in D A cells, with some reaching 15 mV or more. and the durations were typically longer (Fig. 4). ht contrast to the responses observed in D A cells, no rebound depolarizations were observed following the IPSPs elicited in these neurons. Indeed, the IPSP duration extended beyond the latency of the rebound depolarization observed in D A cells. ]"hese striatally evoked IPSPs could also be readily reversed with KCI electrodes (Fig. 4B).

(hAl -075

-IO i

-050 -

-025

0

T . . . . . . .

1

1I Control

Picrotoxin

R,ng=t = 3 6 0 M n

Rmnut = 50.3Mn

re___o = 0.99

r =0.98r)._.¢~ intersection= -75mY

°1._r~_.~...J ,-

~ 0

-40

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(mY)

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Fig. 3. Estimation of the reversal potential of the response elicited in DA cells by systemically administered picrotoxin. Picrotoxin (2.0 mg/kg i.v.) depolarizes the DA neuron (seen as the positive shift of the dashed line crossing the Y-axis) and increases the input resistance (seen as an increase in Mope of the dashed line). The calculated reversal potential of this response, in this ease, was approximately -75 inV.

Responses o f DA and Z R neurons to G A B A agonists G A B A has been shown to inhibit the spontaneous firing of both D A cells and a specific class of Z R cells (identification described in Materials and Methods). F u r t h e r m o r e . in agreement with our previous reports'~L this class of Z R cells is much more sensitive to inhibition by G A B A than are the D A cells (Fig. 5). This differential sensitivity comes into play when a G A B A agonist such as muscimol is given systemically. Thus, systemic administration of the G A B A agonist, muscimol, will increase D A cell firing rate by inhibiting this inhibitory interneuron 40. In an analogous manner, stimulation of the striatum with single pulses of current leads to an IPSP in all D A and Z R cells examined. H o w e v e r , long trains of stimulation were found to lead to increases in D A cell firing. Furthermore, the rebound depolarization occurring in

277 A

B

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40

60

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10

mSeo

Fig. 4. Comparison of IPSPs elicited in DA and zona rcticulata (ZR) cells by striatal stimulation. A: reversal of the striataliy generated IPSP in a DA ncuron resulting from intracellular injection of chloride ions, as in Fig. 2. B: in a similar manner, stimulation of the striaturn (arrow) results in an IPSP in this ZR neuron, which demonstrates the same latency and reversal by chloride injection as the IPSP elicited in DA cells. One exception, however, is that the IPSP elicited in the ZR cells is larger in amplitude and longer in duration. C: overlay of the IPSP elicited in a DA cell versus the IPSP elicited in a ZR cell. Both IPSPs are of similar latency, although the ZR cell appears to have a superimposed early excitatory component to the response. The IPSPs occurring in all ZR cells tested wcrc consistently larger in amplitude and longer in duration than those elicited in DA neurons. Furthermore, the late components of the IPSP in the ZR cell are associated with depolarization of the DA cell. D: striatal stimulation (arrov,') during intraccllular recording from a DA neuron elicits an antidromically activated IS spike superimposed on an IPSP. In many cases, this is followcd by a rebound depolarization sufficient to trigger action potentials (5 repetitions).

D A cells c o r r e s p o n d s t e m p o r a l l y to the late c o m p o -

ing is that D A n e u r o n s m a y i n d e e d be excited bv

nents of the IPSPs in reticulata n e u r o n s . C o u l d this

striatal stimulation in a m a n n e r similar to their exci-

be o c c u r r i n g in a m a n n e r a n a l o g o u s to that of muscim o l - i n d u c e d c h a n g e s in D A n e u r o n activity - - i.e.

tation by m u s c i m o l : i.e. by inhibition of a m o r e

by p r e f e r e n t i a l inhibition of an i n h i b i t o r y i n t e r n e u -

pothesis is c o r r e c t , then i n a c t i v a t i o n of the inhibitory

ron located in the s u b s t a n t i a nigra? This possibility

Z R n e u r o n should cause the e x c i t a t o r y r e s p o n s e of

was tested in the next series of e x p e r i m e n t s .

D A cells to striatal stimulation to r e v e r s e into an

G A B A - s e n s i t i v e inhibitory Z R n e u r o n . If this hy-

inhibitory r e s p o n s e , reflecting the r e m a i n i n g direct DA and non-DA nigral neuron interaction during striatal stimulation

G A B A e r g i c striatonigral D A cell input. T o test this p r e d i c t i o n , m u s c i m o l was a d m i n i s t e r e d in a dose suf-

T h e s a m e m a g n i t u d e of striatal s t i m u l a t i o n that

ficient to totally inhibit the firing of the Z R n e u r o n

p r o d u c e d excitation of D A cells caused a significant

(i.e. 7 m g / k g m u s c i m o l i.v.). T o control for the respi-

d e c r e a s e in the firing rate of the i n h i b i t o r y Z R inter-

ratory d e p r e s s i o n associated with such large doses of

neurons d e s c r i b e d a b o v e (76.2% + 2 0 . 5 % d e c r e a s e in rate, n = 16: Fig. 6). T h e implication of this find-

m u s c i m o l , the rats w e r e p a r a l y z e d in a d d i t i o n to being a n e s t h e t i z e d , and w e r e r e s p i r e d artificially. Un-

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Fig. 5. Ratemetcr recordings of the responses of DA cells (A) and Z R cells (B) to intravenous administration of the G A B A agonist, muscimol (1) and iontophoretically administered G A B A (2). A~: DA cells demonstrate a dose-dependent increase in firing rate in response to systemic administration of the G A B A agonist muscimol (given at arrows). Az: in contrast, directly administered G A B A inhibits the firing of DA cells. Horizontal bars indicate duration of ejection. Numbers above bars specify the ejection current in nA. B~: unlike the DA cells, Z R cells demonstrate a dose-dependent inhibition to systemically administered muscimol. B2: directly administered G A B A will also inhibit Z R cell firing, but at much lower doses than are required to inhibit the DA cells. This differential sensitivity has also been shown for inhibition induced by striatal stimulation. (Muscimol doses in both examples in mg/kg: 0.1,0.1,0.2, 0.4, 0.8, 1.6 and 3.2; G A B A concentration is 0.001 M in (I. 1 M NaCI for the thin lines, and 0.01 M in 0.1 M NaCI for the thick lines.)

der these conditions, the same p a r a m e t e r s of striatal stimulation which originally led to increases in D A cell firing rates before muscimol administration now decreased D A cell firing rates (15.7c?~• __ 8.5% decrease in firing rates, P < 0.01, n = 10: Fig. 7).

STIM

STIM

1 MIN

Fig. 6. Effects of striatal stimulation on Z R cell tiring rate. Stimulation of the striatum with relatively low amplitudes of current (20/~A, 10 Hz stimulation at bar) elicits a strong inhibition of ZR cell firing rate.

ZR cell identification In an attempt to identify this class of Z R neurons with respect to their projection site TM, stimulation of the caudate, superior colliculus and thalamus was done. Although stimulation of each area antidromically activated other types of non-dopaminergic neurons within the nigra (caudate: n = 18; superior colliculus: n = 15; thalamus: n = 29), in no case did the antidromically activated neuron possess the characteristics of this class of zona reticulata neurons. In addition, none of the Z R neurons identified by the criteria given above could be antidromically activated from these regions (n = 18). Thus, although there is a direct striatonigral G A B A e r g i c input to D A cells, trains of striatal stimulation apparently preferentially inhibits a m o r e G A B A - s e n s i t i v e Z R interneuron and thus results in a net removal of inhibition from the D A cells.

279 S_ T I M

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Fig. 7. Striatal modulation of DA cell activity. Stimulation of the striatum at relatively high intensities (3011 ,uA at lIXI I lz. first bar) resuits in a small inhibition of D A cell spontaneous activity. Lowering this current to a much lower intensity (20 I~A at 10 Hz, subsequent bars) now results in an increase in the firing rate of the DA cell. Systemic administration of muscimol at a dose sufficient to completely inhibit the Z R neuron (7.0 mg/kg i.v., at arrow) results in the typical increase in DA cell activity followed bv a slow accommodation. Stimulation of the striatum at the same intensities which excited the DA cell prior to muscimol administration now results in a decrease in firing rate. presumably by a direct effect. Apomorphinc (50 ,ug, kg i.v., arrow) and halopcridol (0.1 mg:kg i.v., arrow) exerted their characteristic effects on DA cell firing rates (i.e. inhibition and reversal of inhibition). Rats administered high doses of muscimol wcrc respired artificially to control for thc depression of breathing produced by this drug.

DISCUSSION

The data presented here suggest that the striatum exerts two opposing influences on D A cell firing: (1) a direct inhibition; and (2) an indirect disinhibition.

Direct effects of striatal stimulation on DA cells The direct inhibition of D A neurons appears to be mediated by the well-characterized striatonigral G A B A e r g i c projection, which is known to directly innervate D A cells. Thus. the latency of the IPSPs elicited in DA neurons by caudate stimulation has a time to onset which corresponds roughly to the fastest reported conduction velocity of antidromically activated striatonigral neurons (i.e., 2.0 ms~l). It is not apparent why this corresponds only to the fastest conduction velocities reported for the antidromically activated striatal cells, although it may have to do with the difficulty reported in reliably activating striatal neurons antidromically from the nigra3",s,z. The IPSP is most likely G A B A mediated, since: (1) there is a well-characterized striatonigral G A B A e r g i c projection: (2) the reversal potential '~ of the IPSP elicited by caudate stimulation was found to lie ~'ithin the range expected for reversal of G A B A e r g i c effects -~,2-s.s4 which are typically mediated by chloride ion conductances in the nervous system4,~,2-s,~,7.~l; (3) intracellular chloride injection was able to reverse the IPSP-'I: and (4) the reversal

potential of the functional G A B A blocker picrotoxin was similar to the reversal potential of the IPSP.

Indirect effects of striatal stimulation on DA cells We reported previously that a specific class of neurons in the zona reticulata (referred to here as Z R cells) directly inhibits D A neurons, and are more sensitive to inhibition by G A B A iontophoresis than are D A cells 4°. Stimulation of the striatum elicits an I PSP in D A cells as well as in zona reticulata neurons. Comparison of the responses of D A and reticulatz~ cells to striatal stimulation suggests that thcy may bc mediated by collaterals of the same striatonigral neurons, since the latency, reversal potential, and chloride reversal of both 1PSPs arc so similar. However, the possibility that they represent two similar but independent projections from the striatum cannot be ruled out by our experiments. The IPSPs produced in Z R cells are much larger in amplitude and longer in duration than those observed in DA cells. This finding correlates well with the known greater sensitivity of Z R cells to directly applied G A B A 4°. Furthermore, low intensity stimulation of the striatum at frequencies sufficient to inhibit Z R cells causes an increase in the firing rates of DA cells. Although there is a striatonigral substance P projection ~.~3-~5, the effccts described here are most likely not mediated by substance P, due to the lack of electrophysiological action of substance P on identified nigral D A cells 2 (Grace, Chiodo and Bun-

28O ncy, unpublished observations) anti the lack of sub-

nigral neurons

stance P receptors in this brain region ~'. l h u s , caudate stimulation appears to affect D A cells m a man-

be blocked by kainic acid lesions t)l ill, -,lrlatunY"

23.2'~.~°,'q.3¢~,''~.'~''~,

an cllct:i

~,~,hlch C;lli

ner similar to that seen with systemicall) adminis-

and by the systemic administration of the ¢ ;AB: x, antagonist, picrotoxin >. In addition, it ha, t)ceu sho~ n

tered G A B A agonists, such as muscimol: both ma-

that the G A B A c r g i c projecnons from the striatum

nipulations act by preferential inhibition of inhibitor} Z R neurons, and thus release the D A cells from inhi-

synapse mainly in the zona reticulata region of the nigra 3.49,577°,73. Furthermore, both I)A blockade alld

bition (Fig. 8). Although we have no direct evidence. these nigral Z R cells arc most likely interneurons.

interruption of the striatonigral loop re~,ults m

since: (1) the G A B A - s e n s i t i v c Z R neurons x,,e have

G A B A e r g i c supersensitivity in zon,, rcticulata cells 22.:'~.,~.,~'~but not in DA cells ":-~,~' l)t~,~ mg iron1

studied are located just ventral to the zona compacta,

our model, the lack of I)A cell supcrscnsitivity to

and a short-axon i n t e r n e u r o n has bccn described in Golgi studies to lie just ventral to I_)A cells and syn-

G A B A could result from the offsetting influence oI: the increase in firing rate of the G A B A c r g t c zona rc-

apse extensively with the D A cells .~ ~,~.sa.,,,~.-~~,: :rod (2) we were unablc to antidromically activate this

ticulata interneuron secondary to interruption of this inhibitory striatonigral G A B A projection This in-

class of Z R ncurons from the commonly described projection sites of nigral I)A and n o n - l ) A neurons -s.

crease in firing rate of Z R cells could tilcrcforc release sufficient G A B A onto the I)A cell,, to prevent the development of I)A cell ( i A B A supcr~,cnsitivity arising from the loss of the striatal G A B A input, kur-

Functional significance A variety of studies support our hypothesis that striatal activity may preferentially inhibit Z R cells and thereby disinhibit D A neurons. Thus. striatal stimulation has been reported to inhibit a n u m b e r of

thor support for a G A B A - ( i A B A - I ) A

loop contcs

from the recent report that administration ol acetylcholine into the striatum leads to incrcase~, in G A B A levels in the zona reticulata, but decrease,, ( ; A B A re-

\~

ZC

/

ZR

FROM STRIATUM

Fig. 8. Partial schematic drawing of proposcd interconnections of striatal projections and nigral neurons. Striatonigral GABAergic fibers are shown here to innervate ZR interneurons and projection neurons as well as DA cells. However. due to the greater sensitivity of the ZR cells to GABA, stimulation of the striatum will preferentially inhibit ZR cells. This will cause a net decreasc in GABA inhibition of the DA cells, and cause them to fire taster. This is analogous to the disinhibition of DA cells produced by administering GABA agonists systcmicall.v%

281 lease in the zona compacta region 6. H o w e v e r , despite this evidence for a G A B A - m e d i a t e d inhibition of D A cells by Z R cells, it must be noted that our data cannot distinguish G A B A e r g i c from glycine-mediated effects. As suggested by others 20, glycine mediation p r o b a b l y should be considered as well, given the significant amount of 6-hydroxydopamine-sensitive strychnine binding r e p o r t e d in the rat substantia nigra 27. The existence of a functional G A B A - G A B A - D A loop may also explain some of the effects of indirectly acting D A agonists on D A cell firing. Thus, a m p h e t a mine is known to inhibit D A cell firing when administered systemically (but not iontophoretically) presumably due to its activation of feedback inhibition secondary to striatal D A release 1,1°.1s. This inhibition is a t t e n u a t e d by lesioning the striatonigral projection l°Al-13A6,17. A m p h e t a m i n e inhibition can be reversed by the G A B A antagonist picrotoxin as well as the G A B A - p o t e n t i a t i n g drug, diazepaml~. The fact that both G A B A antagonists and G A B A - p o t e n t i a t ing drugs reduce a m p h e t a m i n e - i n d u c e d feedback inhibition of D A neurons is paradoxical unless one assumes that each acts via the Z R cell: d i a z e p a m acting by preferentially increasing G A B A - m e d i a t e d striatal inhibition of the Z R cell and picrotoxin by blocking

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