Neuroscience Vol. 44, No. 3, pp. 571 583, 1991 Printed in Great Britain

0306-4522/91 $3.00+ 0.00 Pergamon Press plc ~) 1991 IBRO

F U N C T I O N A L C O N N E C T I O N S OF THE RAT M E D I A L C O R T E X A N D BASAL FOREBRAIN: A N IN VIVO INTRACELLULAR STUDY T. D. WHITE,*~'~A. M. TAN* a n d D. M. FINCH*§ *Brain Research Institute, ]Department of Biology and §Department of Neurology, Reed Neurological Research Center, University of California, Los Angeles, CA 90024, U.S.A. Abstract--Projections between the medial cortex and basal forebrain in the rat were demonstrated by intracellular recordings and the anterograde tracer Phaseolus vulgaris leucoagglutinin. Direct projections between these areas were indicated by antidromic action potentials, short latency ( < 5 ms) orthodromic potentials, and labeled axon terminals in the basal forebrain subsequent to iontophoresis of Phaseolus vulgaris leucoagglutinin into posterior cingulate cortex. High proportions of antidromic action potentials were encountered in responsive cortical neurons (66%) and basal forebrain neurons (97%). Antidromic latencies recorded in the basal forebrain ( < 1.0 ms) revealed fast ascending projections; cortical neurons showed both fast and slow descending projections (latencies of 0.3-3.7 ms). Relatively few synaptic potentials (none in the diagonal band of Broca) and sparse labeling of axon terminals observed in the basal forebrain indicated that the ascending projections may be the more physiologically important or, at least, densest pathway. Polysynaptic feedforward pathways were suggested through long latency ( > 20 ms) inhibitory and excitatory postsynaptic potentials, the former being the more common response. Candidate inhibitory neurons were identified in both cortex and basal forebrain. Possible monosynaptic ( < 5 ms) inhibitory postsynaptic and antidromic responses in these cells provided evidence that candidate inhibitory neurons participate in the reciprocal pathways.

Intense interest in the nuclei of the basal forebrain (BF) has p r o v i d e d a n a t o m i c a l evidence t h a t B F neurons project extensively to the entire cortical mantle. 4'5'2°'2324'27'34'44'47'53 O t h e r a n a t o m i c a l studies have s h o w n t h a t target cortical areas often project reciprocally back to the BF, especially in the rat 17"35'44 a n d to a lesser extent in monkeys. 35'42 In the cat, a n a t o m i c a l 4~ a n d electrophysiologica118'~9 evidence has d e m o n s t r a t e d t h a t medial frontal cortical neurons (including areas 24 a n d 32) project to the preoptic region, diagonal b a n d o f Broca, a n d the h y p o t h a l a mus; the latter two target nuclei have been s h o w n to project back to a n t e r i o r cingulate cortex. 37 The cingulate cortex is one cortical target area whose general cellular characteristics have been studied, 12'45'49 but for which intracellular physiological d a t a c o n c e r n i n g relations with the BF are limited; Y a n g a n d M o g e n s o n 54 have s h o w n t h a t units in the prefrontal cortex can be driven by B F stimulation a n d t h a t acetylcholine ( A C h ) mediates t h a t response. Several studies have characterized the responses of BF n e u r o n s antidromically driven by neocortical or fimbrial stimulation, 2'3'7's'26"28'4°'48 b u t n o n e have

d e m o n s t r a t e d the properties of synaptic responses of B F n e u r o n s driven by cortical stimulation. The present p a p e r reports the reciprocal a n t i d r o m i c a n d synaptic responses o f non-deafferented cells in the medial cortex a n d selected B F nuclei recorded in the brains of intact rats.

:~To whom correspondence should be addressed at: Brain Research Institute, 73-364 Center for the Health Sciences, University of California, Los Angeles, CA 90024, U.S.A. Abbreviations: ACh, acetylcholine; BF, basal forebrain; EPSP, excitatory postsynaptic potential; IPSP, inhibitory postsynaptic potential; PHA-L, Phaseolus vulgaris leucoagglutinin. 571

EXPERIMENTAL PROCEDURES

In vivo electrophysiological techniques Standard methods ~2were used to obtain in vivo intracellular recordings from 140 male Sprague-Dawley rats (20~500 g) anesthetized with chloral hydrate (400 mg/kg, i.p., and 0.1~).4 ml, i.m., to supplement). Briefly, recording electrodes were borosilicate micropipettes filled with 1.0 M potassium citrate in saturated Fast Green dye. Recording sites were marked by depositing the Fast Green with 2 5/zA of negative current (90-100/~ A-min, where 1 # A-min equals 1 ~tA passed for 1 rain) or were calculated from microdrive measurements on Nissl-stained brain sections (100/zm thick). One or two bipolar twisted-wire stimulating electrodes (150-#m-diameter wire) were placed stereotaxically and adjusted to produce maximum strength field potentials in saline pipettes (2-3-/zm tips) placed in recording loci. Electrical stimulation was applied with photically isolated currents of 20f~500 #A (in some candidate inhibitory neurons, currents of up to 1500#A were applied in order to evoke a response), pulse durations of 0.2 ms, and frequencies of 0.5~.0/s. Physiological data were recorded on F.M. tape and played back on a digital storage oscilloscope (Nicolet 1049A) for analysis. In instances when synaptic responses were absent or weak, train stimuli (400/s) of up to 20 ms duration were employed to test responsiveness. Train stimulation was also employed to test for high frequency following of possible antidromic action potentials (e.g. Fig. 7). Suspected short latency ( < 1 ms) antidromic

T . D . V~/HITEC[ al.

572

action potentials were routinely tested by reversal of stimulus polarity to eliminate potential stimulus artifacts. Other criteria for accepting antidromic responses included constant latencies, action potentials in the absence of excitatory postsynaptic potentials (EPSPs) and, when possible, collision (e.g. Fig. 5) of antidromic action potentials with spontaneous action potentials. 3° Response latencies were measured from train onset. If the stimulus artifacts obliterated response latencies, then the latency data were not tabulated. Negative results were not included as nonresponsive cells could not be reliably distinguished from cells damaged by penetration of the recording pipette. Stimulation sites were marked by electrolytic lesions (100/~A for 5 s, both polarities) for the Prussian Blue reaction. Recording sites and corresponding stimulating sites of neurons that exhibited synaptic potentials were summarized in Figs 1 and 2. Locations of recording and stimulation sites of cortical cells that produced antidromic action potentials are presented in Fig. 3. Cortical areas are defined as in Vogt and Peters? ° Coordinates are from the atlas of Paxinos and Watson. 3s As the BF is a functionally and pharmacologically diverse collection of nuclei, we have collected the BF recording and stimulation sites into four groups arbitrarily based upon anatomical and pharmacological criteria. The first group comprised sites within the diagonal band of Broca. The second group included sites within the ventral striatum: ventral pallidum, globus pallidus, and nucleus accumbens. These first two groupings were roughly comparable to sectors Ch2~Ch3 and Ch4, 36 respectively. These nuclei con-

lain large proportions of cholinergic and GABAergic neurons) 3'4~'53 The third group was composed of nuclei lateral and posterior to the medial septum but medial and ventral to the striatum: lateral septum, preoptic area, and lateral hypothalamic area. The third grouping of nuclei was collectively referred to as "intermediate" due to their position between the cholinergic groupings. A fourth group which may have also included cholinergic neurons 1'46 comprised sites within the olfactory tubercle and adjacent olfactory cortex. In addition, the responses of cortical cells were tested by stimulation of the bed nucleus of the stria terminalis.

Anterograde tracing, techniques Micropipettes (0.02 mm i.d.) were used for iontophoresis of 2.5% solutions of Phaseolus vulgaris leucoagglutinin (PHA-L) in 0.5 M sodium phosphate-buffered saline, pIq 7.4) 5 lontophoretic injections (pulsed 5 # A on-off at 15-s intervals for 20 min) were made in posterior cingulate cortex (area 29). Results of PHA-L iontophoretic injections into anterior cingulate cortex (area 24) are presented elsewhere. 5j The animals were allowed to survive 11 days before intracardial perfusion with saline, followed by 4% paraformaldehyde (dissolved in Na-phosphate-buffered saline). The brains were left overnight in 20% sucrose. Frozen sections (50 #m) were incubated in normal rabbit serum and goat anti-PHA-L. Subsequently, biotinylated anti-goat IgG was added. The antigen-antibody complex was visualized through avidin-biotin horseradish peroxidase immunochemistry (Fig. 4).

Synaptic Responses • RECORD

*STIMULATE

~

~B-3.3

.2 1.3

Fig. 1. Location of recording and stimulation sites of cortical cells showing synaptic potentials. Cortical neurons (circles) and their corresponding stimulation sites (asterisks) are indicated by the same number. Neurons stimulated from two different placements are indicated by A and B. Diamonds indicate cells that responded differently to each of two different stimulation sites (cells nos 6, 15). Responses of cell no. 6 are in Fig. 8. Cell no. 15 produced an inhibitory postsynaptic potential (IPSP) following stimulation of the lateral preoptic area and an EPSP and IPSP following ventral pallidum stimulation. Stimulation site numbers separated by a semicolon indicate separate, superimposed placements. Open circles indicate neurons that produced EPSPs. Closed circles indicate neurons that produced IPSPs. Half-closed circles indicate neurons that produced both IPSPs and EPSPs. Frontal sections in this and other figures are based on the atlas of Paxinos and Watson. 3s

Connections of the medial cortex and basal forebrain

573

Synaptic Responses • RECORD

eSTIMULATE

t.2

\ B -0.3

9,10 ~

Fig. 2. Localization of recording and stimulation sites of BF cells showing synaptic potentials. Symbols are as defined in Fig. 1. These cells also exhibited antidromic action potentials.

RESULTS

Neuronal recordings

Recordings were obtained from 72 cortical neurons from 86 rats. Depending on the amplitude of the action potential, a cell was classified as either intracellular ( > 4 0 mV) based on the criterion used by Kandel et al. 21 or "quasi-intracellular" (20-39.9 mV). Criteria for accepting neurons included a 2 0 m V m i n i m u m amplitude of the action potentials, nonfluctuating resting membrane potentials, and sufficient longevity to ensure repeatable, reliable observations. Some of the quasi-intracellular recordings may represent dendritic penetrations 3~'52 or partial penetrations of the cell membrane. There were no significant differences in spike duration, spontaneous rate, or conduction velocities of antidromic action potentials between quasi-intracellular and intracellular neurons. These neurons were regarded as "principal cells" of the cortex and assumed to be pyramidal cells. This assumption was based upon the criteria for identifying principal cells established by McCormick et al. 3~ and by intracellular horseradish peroxidase injections into posterior cingulate cortex? ~These cells

typically exhibited depolarizing postspike afterpotentials [i.e. the descending phase of the action potential (39 _+ 11 mV) did not overshoot baseline but stayed relatively depolarized] and action potential durations at one-half amplitude of 0.6 2.5 ms (1.4 _+ 0.8 ms). Resting potentials were 3 8 + 14mV; spontaneous rates were 15 +_ 14 spikes/s. Recordings were obtained from 34 non-bursting BF cells in 54 rats. Criteria for accepting recordings of neurons were as described above. BF neurons tended to exhibit hyperpolarizing postspike afterpotentials (Fig. 5C) and significantly different action potential durations (0.8 +_ 0.3 ms), rates of spontaneous activity (21 +_ 18 spikes/s), and conduction velocities (14 _+ 7 m/s) of antidromic action potentials (Student's t-test, P < 0.001) as compared to cortical neurons. The action potential amplitudes of BF neurons were 31 + 8 mV; the resting potentials were 35 _+ 12 inV. Projections f r o m the basal Jorebrain to the medial cortex

Antidromic action potentials'. Cells in the BF were antidromically driven by stimulation of all areas of

Y,

~STIMULATE

qg, 3. Localization o f recording and stimulation sites o f cortical cells that exhibited antidromic action potentials. Labels are as defined in Fig. 1,

,RECORD

~,ntidromic Responses

.3

575

Connections of the medial cortex and basal forebrain

A

B Terminal Field

PHA-L Injection Site

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t x

29c~

I t t

-

'

~' k '~

',, ~ " ~

2

Posterior Cingulate Cortex B -5.8

Diagonal Band B 0.2

Fig. 4. Results from an experiment with the anterograde tracer PHA-L showing direct projections from the posterior cingulate cortex to the diagonal band of Broca. The region indicated in B1 by an arrow is shown in photomontage in B2. Scale bar = 20/~m.

the medial cortex tested (Table 1). Of the 34 BF cells found to be responsive to medial cortex stimulation, all exhibited antidromic action potentials (Fig. 5). With few exceptions, antidromic latencies were < l . 0 m s . Mean ( ± S . D . ) antidromic latency was 0 . 6 + 0 . 2 m s (range 0.4-1.1ms). Conduction velocities of antidromic action potentials varied from 5.6 to 28.3 m/s (n = 27). Lengths of neural pathways were calculated according to the medial pathway; ~3

those neurons whose pathway to the cortex was ambiguous were excluded. Synaptic responses in medial cortex. In medial cortex, virtually all recording sites showed synaptic potentials (Table 2). The predominant responses were inhibitory postsynaptic potentials (IPSPs) (50.0% of the responsive cells). IPSP latencies were frequently > 20 ms (32.7 + 28.7 ms) (Fig. 6). Three cells exhibited short latency ( 3 . 6 4 . 8 ms) IPSPs. IPSP durations

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Fig. 5. Examples o f antidromic r c s p 0 r ~ s recorded intraceltularly from ceils in the BF. Column A shows antidromicatly elicited action: potentials. Column B shows the stimulus artifact onlydem0nstrating the all-or-none properties of these potentials. Note that B2 and B3 show possible collision o f spontaneous action potentials with antidromie action potentials. Column C shows spontaneous ~ n potentials with hyperpolarizing postspike afterpotentials. Stimulation artifacts (500 gA) in this and other figures have been truncated. were p r o l o n g e d (101 + 61 ms), M e a n I P S P a m p l i t u d e w a s - 4 . 2 + 2.6 inV. T h e r e w a s n o o b s e r v e d relationship b e t w e e n I P S P a m p l i t u d e a n d m e m b r a n e resting potential. E P S P s were i n f r e q u e n t l y o b s e r v e d (12.8%). E P S P latencies (79.6 + 74.1 ms) a n d d u r a t i o n s

(144 _.+ 178 m s ) were relatively long. E P S P d u r a t i o n s were o f t e n t r u n c a t e d w h e n a c c o m p a n i e d by a n I P S P (Fig. 6). A s h o r t l a t e n c y ( 4 . 4 m s ) p o s s i b l y m o n o s y n a p t i c E P S P was p r o d u c e d in a n area 4 n e u r o n f r o m s t i m u l a t i o n o f the b e d nucleus o f the stria terminalis. M e a n E P S P a m p l i t u d e was 6.5 + 4.9 mV.

Table 1. Response latencies of basal forebrain cells to stimulation of the medial cortex Antidromic Stim. site 24

32

29

17/18 Totals

Record site DBB I. NUC. V. STR. OT DBB I. NUC. V. STR. OT DBB I. NUC. V. STR. OT DBB

n

n

Mean _.+S.D.

Range

3 4 8 4 1 1 1 1 2 6 2 3 3 39

3 4 7 4 1 1 1 1 2 6 2 3 3 38

0.4 0.1 0.6 0.1 0.6 0.2 0.7 0.1 0.7 . 0.6 . 0.4 . 0.7 . 0.6 0.2 0.5 0.1 0.6 0.1 1.0 0.2 0.5 0.1 (97.4%)

0.4-0.6 0.5-0.8 0.4-t.0 0.64).8 . . . . . . . . . 0.5-0.8 0.443.7 0.5-0.7 0.8-1.1 0.4-0.6

EPSP n . .

Mean __+S.D. Range . .

2 1

IPSP ........................ n Mean + S.D. Range

. .

7.1 30.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 (7.7%)

. . 0.7 -. . . .

.

.

6.6-7.6 -. . . .

.

. . .

. .

. . .

. .

. . .

.

.

.

. 25.1 0.1 25.6 6.0 274 -. . . 1 85.0 -. . . . . . . . . . . 1 31.7 -. . . . . . . 10 (25.6%) 3 4 1

25.0--25.2 16.8-30.5 -~

--

Neurons that responded with antidromic action potentials to two stimulation sites are reported as independent events. Response latencies in ms. DBB, diagonal band o f Broca; I. NUC., intermediate nuclei; OT, olfactory tubercle; V. STR., ventral striatum.

577

Connections of the medial cortex and basal forebrain Table 2. Response latencies of medial cortical cells to stimulation of the basal forebrain EPSP latency

Antidromic latency Stim. site

Record site

DBB

29d,V 29c,V 29b,V 4,V 4,I1/I11" 24b,V 24a,III 24a,V Intermediate nuclei 4,III 4,V 24b,II 24b,V/VI 24a,I1 24a,Vl Ventral striatum 4,V 24b,V/VI 24a,V OT

BNST

Totals

n

n

4 6 1 8 8 10 4 3

3 6 1 2 8 9 4

Mean + S.D. 0.4 0.5 0.5 0.4 0.7 0.5 0.5 --

0.1 0.2 . -0.1 0.1 0.1 --

1 7 1 4 1 1

1 2

3.2 0.6

Range

n

0.44).5 0.34).8 . . 0.4 0,54).9 0.44).7 0.4-0.6

2 .

1

2.1

-0.2 . 0.2 . .

3 5 1

2 4 1

2.2 0.6 0.8

1.8 0.9 3.5 0.2 0.44).8 . . . .

4,V 24b,V 24a,V

4 2 l

1 2

0.5 0.6

-0,3

4,V 24b,li 24b,V/VI 24a,II1 10,V/VI

3 1 5 1 2 86

.

.

.

2 .

0.8 .

.

.

. 2 1 4 1

. 57

.

1.8 0.7 3.7 . 1.1 0.5 0.7 . . . . (66.3%)

Mean + S.D.

-60.9 11.3 . . 13.9

1 1

0.6-1.0

. 1

.

.

.

-0.44).8 . . 1.3 2.3 . . 0.4 1.6 . .

Range

--. . --

.

.

. . .

.

. . .

.

. .

1

--

4.4 . --

. .

. .

1 92.3 . . . . . . . .

-. 1 11

n

64.8 70.9 14.7 115 --. . . . . . . . . . . 125 -. . . . . 147 138 49.3 244

. . . 1 . 2

0.54).8

IPSP latency

--

--.

145 -(12.8%)

--

Mean + S.D.

3 2 . 7

81.8 26.4 . 35.9 . . 4 37.9 . . 1 22.8

25.1 178 3.6 49.3

18.8

9.1~52.5

19.4

23.8 66.2

9.4

8.4 29.9

1 4 . 3 1 1

21.8 50.5 12.0

14.8

11.4 38.8 --

2 I 1

24.4 6.3 4.1

9.4

17.8 31.1

2 1 1

32.8 35.2 68.6

2.2

31.2 34.4

3 1 1 1 2 43

4.8 18.6

Range

83.8 32.3

21.2 7.6 9.9 26.3 67.5 23.0 48.9 -23.1 1.6 22.0 24.1 (50.0%)

Neurons that responded with antidromic action potentials to two stimulation sites are reported as independent events. Response latencies in ms. DBB, diagonal band of Broca; BNST, bed nucleus of the stria terminalis: OT, olfactory tubercle. *Contralateral stimulation.

Projections from medial cortex to the basal forebrain Anterograde tracing. In o r d e r to c o n f i r m m e d i a l cortical e f f e r e n t s to B F , t h e a n t e r o g r a d e t r a c e r P H A -

°

2 0 0 MSEC Fig. 6. Response of a principal cell in layer 11 of area 24a to stimulation (arrow) of the lateral septum showing an EPSP (13.9ms latency) with action potentials and IPSP (50.5 ms latency) with postinhibitory excitation. Electrical stimulation was with 0.2-ms pulses of 500 # A intensity. (A) Single trace. (B) Average of 10 traces.

L w a s injected i n t o a r e a 29. H i g h c o n c e n t r a t i o n s o f s u b c o r t i c a l a x o n t e r m i n a l s were o b s e r v e d in ipsilateral c a u d a t e - p u t a m e n , c l a u s t r u m , a n d t h e reticular, anteromedial, laterodorsal, interanteromedial, and b o t h ipsilateral a n d c o n t r a l a t e r a l a n t e r o v e n t r a l t h a l a m i c nuclei. O t h e r m o r e s p a r s e p o s t e r i o r cortical e f f e r e n t s were o b s e r v e d in t h e d i a g o n a l b a n d o f B r o c a (see Fig. 4), m e d i a l s e p t u m , o l f a c t o r y t u b e r c l e , lateral p r e o p t i c a r e a , a n d t h e stria t e r m i n a l i s . Antidromic action potentials'. O f t h e 72 cells in t h e m e d i a l c o r t e x t h a t r e s p o n d e d to B F s t i m u l a t i o n , 48 (66.7%) produced antidromic action potentials. Test s t i m u l a t i o n s o f t w o B F sites were a t t e m p t e d in 25 n e u r o n s a n d six cells c o u l d t h u s be a c t i v a t e d (Fig. 3). T h e m e a n a n t i d r o m i c l a t e n c y w a s 0.8 + 0.7 m s ( r a n g e 0 . 3 - 3 . 7 m s ) ( T a b l e 2). T h e s e l a t e n c i e s c o r r e s p o n d e d to c o n d u c t i o n velocities o f 1 . 9 - 4 5 . 0 m / s (I 7 ± 9 m / s , n = 47). E i g h t cells in c o n t r a l a t e r a l superficial layers o f a r e a 4 s h o w e d a n t i d r o m i c a c t i o n p o t e n t i a l s following diagonal band of Broca stimulation. Synaptie responses in basal forebrain. R e l a t i v e l y few B F cells ( 2 9 . 4 % o f r e s p o n s i v e n e u r o n s ) s h o w e d synaptic potentials following stimulation of medial c o r t e x ( T a b l e 1). I n h i b i t o r y s y n a p t i c p o t e n t i a l s o f l o n g l a t e n c y ( 1 6 . 8 - 2 7 4 m s ) were r e c o r d e d f r o m cells ( F i g s 7, 8) in t h e v e n t r a l s t r i a t u m , lateral s e p t u m , l a t e r a l p r e o p t i c a r e a , lateral h y p o t h a l a m i c area, a n d t h e o l f a c t o r y t u b e r c l e . O n e cell in t h e v e n t r a l s t r i a t u m

T, D. WHITEet al.

578

.& %

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I 20mV

i

100 mm~

B Fig. 7. Response of a non-bursting cell in the lateral septum to stimulation (arrow) of area 32 showing a long latency (85 ms) IPSP and short latency antidromic action potentials with possible loss of somatic-dendritic spikes within the train stimulus (500 #A). (A) Single trace. (B) Average of 12 traces. produced IPSPs in response to both anterior (area 24) and posterior (area 29) cortical stimulation; however, no other posterior cortical stimulation site produced a synaptic potential in BF non-bursting neurons. Durations of IPSPs averaged 99.2 + 76.5 ms. Mean IPSP amplitude was - 3 . 5 + 1.8 mV. No relationship was observed between IPSP amplitude and membrane resting potential. Three (7.7%) EPSPs were observed. All were produced in ventral striatum or olfactory tubercle cells by stimulation of area 24. In all cases, EPSPs were of

A

short duration (13.7 _ 3.7 ms) and followed by IPSPs (Fig. 8). Mean EPSP amplitude was 4.5 _+ 2.6mV.

Responses of candidate inhibitory cells Distinctive classes of neurons, regarded here as candidate inhibitory neurons, were identified in both cortex and BF. Respectively, these cells were similar to "fast-spiking" cells identified in neocortex 33 and hippocampus 22"22a and "bursting" cells in the medial septum--diagonal band of Broca complex.16 These cells typically displayed hyperpolar-

B

I 2OnlY

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Fig. 8. Response of a non-bursting cell in the globus pallidus to stimulation (arrow) of area 24 showing an antidromic action potential, EPSP (latency of 7.6 ms), and IPSP. Antidromic spikes are superimposed on the stimulus artifact (500/~A), and therefore appear smaller than spontaneous action potentials. (AI) Single trace. (B1) Average of five traces. (A2) Single trace at higher time resolution showing antidromic action potential and EPSP with triggered action potential. (]32) Single trace showing antidromic action potential and EPSP without triggered action potential.

Connections of the medial cortex and basal forebrain

579

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A

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600

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Fig. 9. Responses of candidate inhibitory neurons. Column A shows single traces of a cell (vertical limb of the diagonal band of Broca; stimulus current = 500 #A) that responded with short duration bursts of action potentials followed by inhibition (AI) and a cell (lateral preoptic area; A2 stimulus current 500 1000/~A, A3 stimulus current 100~1500/~A) that produced long duration bursts of action potentials without inhibition (A2, 3). Column B shows frequency histograms of action potential latencies produced by the cells in column A. B1 and B2 show the action potentials of eight traces. B3 shows the action potentials of six traces, x-Axis of histograms is in ms; y-axis is number of spikes/bin (20-ms bin width).

izing postspike afterpotentials, short half-height amplitude action potential durations ( 0 . 4 + 0 . 1 ms, range 0.34).6ms), and responded to depolarizing synaptic potentials with high frequency bursts (180+_ 140Hz) of action potentials. Mean spontaneous firing rate was 29 +_ 21 Hz. Such cells were difficult to penetrate and, as a result, often rendered action potentials of < 2 0 inV. It is probable that a portion of these cells represent extracellular recordings. Our sample of candidate inhibitory neurons in the BF was probably not exhaustive; putative nonbursting G A B A e r g i c neurons 16 could not be positively identified. Of the cortical (n = 79) and BF (n = 38) cells that responded to stimulation, seven (8.9%) and four (10.5%), respectively, were identified as candidate inhibitory neurons. Seven of the total of 11 neurons responded with bursts of action potentials to stimulation. As reported elsewhereJ ] cortical candidate inhibitory neurons exhibited short duration stimulus-

evoked bursts of action potentials (16.7_+4.6ms, n = 6) followed by inhibition (Fig. 9, row 1) or prolonged bursts (213 +_ 11.5 ms, n = 3) without evidence of inhibition (Fig. 9, rows 2, 3). One neuron ( F R I 1, cell 1) exhibited a short duration burst without inhibition in response to ventral pallidum stimulation. Three cortical candidate inhibitory neurons in which no excitatory burst was observed exhibited antidromic action potentials (latency 0.5 _+ 0.2 ms). Mean conduction velocities of these neurons was 27 _+ 16 m/s (range 21-45 m/s). Two of the latter cells produced IPSPs (Fig. 10). Inhibition when present was of similar duration (187 _+ 104 ms, n = 7) to that observed in non-bursting or principal cells. Synaptic potential latencies were generally long; however, one short latency EPSP (FR18, cell 3) and one short latency IPSP (DC19, cell 1) were found. Synaptic potential amplitudes were not reported as probable extracellular recordings were included in the sample.

580

T D. WHITE et al.

A

B

40 mV

2L

J

30 mV

2OO E C

1.0

MSEC

Fig. 10. Response of a fast spiking cell (candidate inhibitory neuron) in layer V of area 29b to stimulation (arrow) of the diagonal band of Broca showing an antidromic action potential and IPSP (latency of 8.7 ms) with postinhibitory excitation. (Al) Single trace. Electrical stimulation was with trains of 430/s for l0 ms with 0.2 ms pulses of 500#A intensity. (A2) Average of six traces showing IPSP and postinhibitory excitation. Electrical stimulation was with trains of 430/s for 10 ms with 0.2 ms pulses of 200-500 #A intensity. (Bl) Single trace showing antidromic action potential. Electrical stimulation was with 0.2-ms pulses of 200 gtA intensity. (B2) Single trace showing stimulus artifact. DISCUSSION

This study found electrophysiological and anatomical evidence for reciprocal projections between medial cortex and the BF. Synaptic potentials were generally long latency but these projections included a few short latency probable mono- or disynaptic inhibitory efferents as shown by very short latency ( < 5 ms) IPSPs in cortical neurons and antidromically driven candidate inhibitory neurons in the cortex. Higher proportions of antidromicaUy driven BF neurons and synaptic potentials in cortical neurons indicated that the BF projection to medial cortex may be the more physiologically important, or, at least contains a greater number of projection neurons than the reciprocal corticofugal pathway. In spite of the demonstration of a significant number of antidromically driven cells in the cortex, there were few synaptic potentials observed in the BF and none in the diagonal band of Broca. Posterior cortical stimulation sites were especially ineffective in producing synaptic potentials in BF. These results were consistent with the relative paucity of cortical axon terminals in BF nuclei after injection of PHA-L into area 29. In a previous study, 5~ injection of PHA-L into area 24 demonstrated heavy projections to caudate-putamen, claustrum, and the anteroventral and mediodorsal thalamic nuclei. Other sites with sparse axon terminals were the medial septum, diagonal band of Broca, globus pallidus, lateral septum, lateral preoptic area, olfactory tubercle, and bed nucleus of the stria terminalis.

BF efferents to medial cortex project via the medial pathway around the genu of the corpus callosum and distribute posteriorly through layer VI of the cingulate cortices. 24'27 Reciprocally projecting cortical efferents follow similar pathways to areas from which they receive input. '7,~ It follows that our anterior medial cortical and BF stimulating sites may have stimulated fibers of passage rendering high proportions of antidromically activated cells. This may, in part, explain why high proportions of antidromicatly driven cortical cells and few synaptic potentials were found within the BF. The antidromic latencies of BF neurons reported here (mean 0.6 ms) differed from those of extracellular studies 2'3'26'28'4°'48 whose means varied between 2.3-4.8 ms. This discrepancy may be attributed to differences in technique and stimulation site. An in vivo intracellular investigation of septohippocampal neurons 7 also found that intracellular techniques produce significantly shorter antidromic latencies (mean 1.1 ms) than extracellular technique s. This difference was putatively due to electrode selection (only larger cells with faster conduction velocities were penetrated). 7 In addition, cortically projecting BF neurons reportedly exhibit significant terminal branching, z,3 Shorter antidromic latencies produced from deeper stimulation sites suggest that the terminal branches are non-myelinated and/or of thin diameter. 3 The stimulation sites used in the present study (Fig. 2) probably preferentially stimulated the deeper, myelinated portions of axons of cortically projecting

Connections of the medial cortex and basal forebrain BF neurons. These results are consistent with the stimulation of fibres of passage as previously discussed. Moreover, BF neurons with fast conduction velocities and very short antidromic latencies ( < 2 ms) must be accepted considering the short latency ( < 5 ms) synaptic potentials recorded in cortical neurons. In all recording sites, the great majority of synaptic potential latencies were relatively long ( > 15 ms). These results indicated that the observed synaptic potentials could be due to polysynaptic feedforward excitation and inhibition through local circuits, through other nuclei such as the thalamus, 29 and/or to temporal summation of convergent afferents. It is also possible that some long latency responses may reflect very slow conduction velocities of projection neurons or slow activation of second messenger systems. In cortex, feedforward inhibition was suggested by short latency IPSPs (latencies of 5-10ms) in principal cells (n = 3 ) and a possible monosynaptic latency in a candidate inhibitory neuron. The short latency inhibitory responses could also reflect a direct G A B A e r g i c projection from the BF to the cortex which has been recently demonstrated by Fisher et al. ~3 In addition, disinhibition of cortical principal cells is indicated by both possible monosynaptic (3.9ms) and short latency (8.7ms) inhibition of cortical candidate inhibitory neurons (Fig. 10), a mechanism that has been demonstrated for the septohippocampal pathwayJ 4 Evidence for feedforward excitation was seen from long latency EPSPs (Fig. 6). Convergence was evidenced in cortical principal cells by synaptic responses from stimulation of two BF sites. In addition, cortical principal cells and three candidate inhibitory neurons were also

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responsive to parahippocampal stimulation. 51 In the BF, similar feedforward mechanisms (Figs 7, 8), disinhibition (Fig. 9, row 1), and convergence (Fig. 9, rows 2, 3) were also in evidence. These observations are consistent with highly branched terminal fields of projection neurons. 2'3 The medial cortical responses to BF stimulation seen here differed substantially from those evoked by other efferents. Whereas IPSPs were the predominant synaptic response of cortical neurons in this study, the typical responses of cingulate cortical neurons to stimulation of the corpus callosum in t,itro 49 or the subiculum complex and thalamus in vivo t: were short latency EPSPs which may or may not have been accompanied by longer latency IPSPs. In contrast, parahippocampal stimulation 5L produced fewer EPSPs but similar latencies and frequencies of IPSPs in medial cortex. When present, EPSPs of medial cortical neurons of this study were often of very long latency ( > 50 ms, n = 6) and not necessarily due to postinhibitory rebound; only two occurred subsequent to an IPSP. Very long latency EPSPs could be caused by a slowly activating second messenger system such as has been suggested for cholinergically modulated responses of cortical n e u r o n s P 1°25"~2 The possible monosynaptic excitatory responses produced in cortical cells after stimulation of the diagonal band of Broca and ventral striatum were regarded as candidates for direct cholinergic activation; however, inhibition from cholinergic neurons 939 or other cells is also possible. Acknowledgements--This research was supported by the

National Institutes of Health Grant NS 23074. The authors thank Mr Robert Gellibolian for his assistance.

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