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Ventrostriopallidal functional interconnections with cortical and quasi-cortical regions
Brain Research Bulletin, Vol. 37, No. 4. pp. 329-336, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0361-9230/95 $9.50 + .OO
Pergamon 0361-9230(94)00281-9
Ventrostriopallidal Functional Interconnections With Cortical and Quasi-cortical Regions E. I. BARRAGAW AND H. FERREYRA-MOYANO*t’ */nstituto de lnvestigacidn Me’dica, Mercedes y Martin Ferreyra, Cdrdoba, Argentina tC&edra de Psicobiologh Experimental, Escuela de Psicologia-Facultad de Filosof,;3 y Humanidades, Universidad National de Cdrdoba, Cdrdoba, Argentina [Received 27 May 1994; Accepted 25 October 19941 its former boundaries: it is now clear that both the striatum and globus pallidus extend rostroventrally to reach the base of the brain, forming a ventral striatum (VS) and ventral pallidum (VP) [22]. On the basis of specific protein markers, DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein M 32.000) and synaptophysin, a marker for nerve terminals that also effectively labels pallidal synapses (according to Walaas and Ouimet [56]), the VP was traced rostroventrally from the globus pallidus to the superficial layers of the OT, moreover, the VS and the VP were found to be densely intermingled in parts of the OT and medial forebrain regions, and clearly separated in more caudal regions. These results indicate that the ventrostriopallidal region (VSPR), at least in the rat, extends both rostrocaudally and dorsoventrally, in a highly complex, intermingled fashion, throughout most of the basal forebrain [56]. On the other hand, the relationship of the VSPR with olfactory forebrain structures has not been clearly defined. For example, several authors have emphasized its close relationship to the olfactory system proper on the basis that a) some of its components, such as the OT, receive a direct input from the main olfactory bulb (MOB); b) part of the OT, especially its lateral region, fulfills the requirements for being classified as a cortex and, thus, can be considered to be part of the olfactory cortex [33,49]; and c) the entire rostrocaudal extent of the later is mapped onto the OT from lateral to medial [30]. Nevertheless, several investigators have emphasized the fact that this region, in spite of its layered configuration, should be considered as a ventral extension of the basal ganglia [21,23,34,39]. Others, however, seem to view the OT as a transition area between the olfactory cortex and the striatum [33,55]. An alternative way to clarify this controversy would be to functionally analyze putative reciprocal connections with the VSPR from forebrain regions known to be morphologically as well as functionally related to the olfactory system. The present study addresses the organization of descending as well as ascending projections from cortical and quasi-cortical regions to the VSPR in the rat. All of these structures, in addition, have reciprocal mono- or disynaptic connections with the MOB: the anterior olfactory nucleus (AON) [20], the basolateral nucleus of the amygdala (BLA) [13], the OT [1,4], the lateral and ventrolateral orbital cortex in the prefrontal region [10,18,41,51,52],
ABSTRACT: A total of 287 neurons were antidromically driven in quasi-cortical regions, i.e., anterior olfactory nucleus (24%), basolateral amygdala (13%), main olfactory bulb (4%), prefrontal cortex 137%1.and In the hippocampal formation (22%) following macro-’ and’&rostimula& of t6e rat’s ventrostriopallklal & glon (VSPR). In addiion, a substantial number of units @I= 175) were also transynaptiilly affected In all these structures by shocks delivered to the VSPR. Excitatory effects were detected in 50 neurons (56.1% of ntsponslve Cells), 36 cells (40.4%) responding with inhibition of spontaneous discharges. Conversely, stimulation of cortical and quasi-cortical regions antldromlcally discharged (n = 37) or transynaptically affected (n = 151) units in the VSPR; 168 neurons were not responsive to VSPR stimulation. Axon collaterallxation (branching) of 14 neurons in anterior olfactory nucleus, basolateral amygdela, and hippocampal formation was revealed wlth the use of the reciprocal collision test. Conduction properties of 35 neurons, evaluated by paired-pulse stimulation, indicated that only 23% showed a significant increase in conduction velocity and a decrease In threshold during the supernormal phase. The present findings first, described s&g interconnections between the neocorteX and striatal structures, and second, that the VSPR as suggested by previous structural, hodological, and hlstochemlcal stud& seems to maintain a more close relationship with olfactory related structures than hlthetio suspected. KEY WORDS: Basal ganglia, Ventral pallidum and strfatum, Antidromlc unlt drlvlng, Transynaptic effects, Main olfactory bulb, Anterior olfactory nucleus, Basolateral amygdala, Prefmntal cortex, Hlppocampal fotmatlon.
INTRODUCTION The basal forebrain
is a complex structure, which includes, among other regions, the substantia innominata, the basal nucleus of Meynert, the nucleus accumbens (NA), the olfactory tubercle (OT) and cortex (cortex piriformis), and the amygdaloid nuclei. Clinical, physiological, and behavioral studies have stressed the crucial role played by the basal forebrain in behaviors ranging from basic drives and emotions to higher cognitive functions [ 11. One of the basal forebrain components, the striatopallidal system, has recently undergone a considerable reevaluation in relation to
’ Requests for reprints should be addressed to H. Ferreyra-Moyano, INIMEC, Casilla de Correo 389, 5000, C6rdoba. Argentina. 329
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BARRAGAN AND FERREYRA-MOYANO
and the comu Ammonis area 1 (CAI) region and subiculum in the hippocarnpal complex (HP) [54]. Antidromic activation of neurons was used as a reliable method for the topographical localization of cell bodies, provided certain constraints were met, which are described in the Method section. In addition, effects of VSPR stimulation were assessed on unit activity recorded throughout the AON, MOB, prefrontal cortex (PFC), BLA, and HP. A preliminary study has been published [5]. ABBREVIATIONS AON: anterior olfactory nucleus; BLA: basolateral nucleus of the amygdala; CA1 : comu Ammonis area 1; HP: hippocampal formation; MOB: main olfactory bulb; NA: nucleus accumbens; OT: olfactory tubercle; PFC: prefrontal cortex; RCT: reciprocal collision time; VP: ventral pallidum; VS: ventral striatum; VSPR: ventrostriopallidal region. METHOD Sixty-five rats (Wistar modified strain), 200-300 g, were anesthetized with Urethane (1.2 g/kg, IP) and mounted in a stereotaxic instrument with the incisor bar 3.3 mm lower than the ear bars, according to the stereotaxic atlas of Paxinos and Watson [38]. Core temperature was monitored with a digital rectal thermometer and maintained between 37 and 38°C throughout the experimental session. Concentric bipolar stimulating electrodes (Rhodes Med. Instruments, Inc., Model NEX-100, shaft diameter 0.5 mm) were advanced from the brain surface and positioned in the VSPR. Constant current monophasic square wave pulses between 0.1 and 1.3 mA in amplitude and 200 ms in duration were delivered through these electrodes at a rate of 0.2 to 1 Hz. Extracellular anti- and/or orthodromic unit responses were recorded throughout the AON, the MOB, the BLA, the hippocampus, and the PFC following VSPR stimulation. In a number of experiments, single-unit activity was recorded from the VSPR and responses to MOB, AON, BLA, and CA1 region and ventral subiculum stimulation was also evaluated. Extracellular recording of single unit activity and microstimulation was performed with stainless steel microelectrodes (A-M Systems, Inc., 12 Mfl impedance at 1000 Hz), which were advanced from the brain surface by means of an hydraulic microdrive (Narishige MO-8). Single-unit activity was amplified and displayed on a storage oscilloscope as filtered (300- 10 kHz bandpass) signals and monitored with a loudspeaker. Criteria for antidromic unit driving has been extensively discussed in previous articles [l&17]. Briefly, the evoked action potential was considered to arise from a single cell when: a) it had an all-or-none amplitude at threshold levels of stimulation; b) the evoked and spontaneously generated potentials had similar waveforms; c) the spike was able to consistently follow twin pulse stimulation at frequencies of 200 Hz or greater; d) in some cells, the waveform exhibited an initial segment and a somatodendritic component in the second of two closely spaced action potentials; and e) the antidromic spikes collided and were blocked by a spontaneous discharge at a critical time interval equal to twice the antidromic latency plus the absolute refractory period of the axon at the stimulated site. When a neuron was antidromically activated from two distinct sites of stimulation, this could have been due either to stimulation of the same axon at different sites or to stimulation of two collaterals of the same axon. The reciprocal collision test was used to distinguish between these two possibilities. The reciprocal collision time (RCT) corresponds to the maximal interval between the stimuli applied at both sites producing a blockade of the second antidromic spike.
When this RCT was greater than the difference between the latencies of the two antidromic responses plus the refractory period of the second stimulated axonal branch, it was concluded that the neuron was branched. Antidromic latency was measured from stimulus artifact to the base of the spike (first departure from baseline). Initial segment spikes followed by somato-dendritic components were observed in 23% of antidromically invaded cells. When only one component was present, it was assumed to be the somato-dendritic based on the total spike duration and the ratio of positive to negative phase duration. The modal value of ten antidromic latency measurements was assigned as the control latency for a given neuron to eliminate antidromic latency jitter that occurs during periods of spontaneous impulse activity [ 15,501. The estimated conduction distance between recording and stimulating electrodes was calculated by measuring the distance between the shafts of the stimulating and recording electrodes in the in situ preparation. This figure was later adjusted by estimating the distance between the blue spots (Prussian blue reaction) in coronal sections serially cut at 80 pm intervals. To compensate for spread of stimulus current, 0.2 mm was subtracted and this value multiplied by 1.2 to correct for possible nonlinear axonal trajectory, that is, a 20% increase in the distance separating the stimulating and recording sites was allowed to compensate for tortuosity in the axon’s pathway. In calculating estimated axon conduction velocity (CV), conduction time was obtained by subtracting 0.2 ms from the antidromic latency to compensate for the utilization time at the site of stimulation and delay of invasion near the cell body. Neuron excitability was assessed with the help of a threshold hunter. This device was designed and constructed in our laboratory [9], with the purpose of evaluating threshold variations in antidromically activated neurons before, during, and following the supernormal period of increased CV and decreased excitability. The technique is based in the method of constant response [2]: during threshold evaluation, the duration of an electric pulse applied to the neuron’s axon is automatically varied, depending on the presence or not of an action potential. For a given spike, the stimulus amplitude is progressively decremented (- delta i) up to a point where the neuron ceases to respond, and thereafter, the stimulus amplitude is progressively increased (+ delta i) until slightly suprathreshold values are obtained. The procedure guaranties a discharge probability of the neuron equivalent to 50% of all applied stimuli, and the simple monitoring of the stimulus amplitude is enough to obtain the threshold value for a predetermined intensity. For more details, refer to Cinelli and FerreyraMoyano [9]. At the end of the experiment, positive current (lo-20 mA) was passed through the recording and stimulating electrodes, and tip position was later identified as blue spots in frontal-cut sections treated with Potassium Ferrocianide and HCl (l%.)(prUsSian Blue reaction). The tip of the VSPR stimulating electrode was found to lie between 11.2 and 8.6 mm rostral to the interaural plane. The figures of Young et al. (58) were used to precisely match the blue spot in relation to the VS and VP stimulated areas. These included, in addition to the above-mentioned structures, the deep region of the OT, the boundary zone between the VP and NA, the VP and substantia innominata, and the VP and fundus striati. RESULTS Electrical stimulation of the VSPR resulted in antidromic driving of neurons in the AON, the BLA, MOB, PFC, and hip-
BASAL FOREBRAIN INTERCONNECTIONS TABLE 1 ANTIDROMICALLY DRIVEN UNITS FOLLOWING VSPR STIMULATION (n: = 287)
Smtctun
n
Percent of Total
AON BLA HP MOB PFC*
70 37 63 11 106
24 13 22 4 37
LatetlCy Mean+SD
cv m/s Mean 2 SD
8.9 + 13.1 2 14 2 8.3 + 11.7 2
0.6 0.4 0.7 0.9 0.3
4.12 6.25 5.07 3.23 4.28
? 2 + 2 k
0.33 0.16 0.27 0.54 0.18
CV: conduction velocity (m/s). HP: includes cells in CA1 and subiculum. SD: standard deviation. n = number of neurons tested. * Prefrontal region 1 (Frl), n = 4, Prefrontal region 2 (FR), n = 64; Lateral orbital region (LO), n = 15; Ventrolateral orbital region (VLO), n = 23.
FlG. I. A, B, C, D, E, F, G, and H show 23 dorsoventral penetrations of the recording microelectrodes at five different A-P planes [38]. Antidromically invaded cells following electrical stimulation of the VSPR are shown as small horizontal lines. Abbreviatiolrs:amg = basolateral amygdala; aon = anterior olfactory nucleus; cal = comu Ammonis area 1 (hippocampus); ent = entorhinal cortex; s = subiculum; fr = prefrontal cortex.
pocampal structures (CA1 and subiculum). Figure 1 shows 23 of 132 dorso-ventral penetrations of the recording microelectrode made at different antero-posterior forebrain planes; small horizontal lines in each tract indicate location of neurons that responded with an invariant latency and a discrete threshold to VSPR stimulation. Neurons antidromically driven in the MOB were found to lie at the level of the mitral cell layer. Table 1 shows the distribution of these neurons in the prosencephalon as well as some of their electrophysiological characteristics. Estimated CV for all these neurons indicate that they belong to the C-fiber type (CV < 1.3 m/s). Roughly 50% (n = 89) of spontaneously discharging neurons recorded in the AON, BLA, MOB, PFC, and HP were responsive to VSPR stimulation; excitation was present in 56% of responsive units, temporary inhibition of spontaneous discharges was observed in 40% and 4% of cells exhibited an initial period of excitation followed by inhibition (Table 2). A substantial number (45%) of those neurons in AON, BLA, HP, and MOB, which were transynaptically affected following
VSPR stimulation, showed inhibitory effects (Table 2). Inhibition, that is, temporary cessation of spontaneous discharges, was present in either antidromically driven cells when challenged with a conditioning volley to the VSPR or in spontaneously active cells (Fig. 2). Strikingly similar periods of cessation of ongoing spontaneous discharges were seen in units recorded in the AON, CA1 region, subiculum, and amygdala when stimulation was attempted from two different brain regions (Fig. 2). A total of 229 neurons were studied in the HP (CA1 and subiculum). Table 3 summarizes the effects of AON, MOB, PFC, and VSPR stimulation on these neurons. Eighty-one neurons were antidromically discharged following electrical stimulation of these regions; in addition, a large number of spontaneously discharging neurons in the HP exhibited a temporary cessation of spontaneous discharges following AON or VSPR stimulation. In a different experimental setup, effects of electrical stimulation of several prosencephalic structures (AON, MOB, PFC, and HP) were assessed on 356 neurons recorded in the VSPR. Table 4 shows that approximately 53% of neurons were either antidromically driven (n = 37) or were otherwise affected in their spontaneous
discharge
pattern (n = 151).
Branched Axon Neurons Fourteen neurons were identified in the AON (n = 5), BLA (n = 2), CA1 region (n = 3), and ventral subiculum (n = 4) that possessed bifurcated axons, with one branch directed to the TABLE 2 EVOKED RESPONSESOF
AON, BLA, HP, AND PFC UNITS FOLLOWING VSPR STIMULATION (n = 175)
Pattern of Response
AON
BLA
HP
MOB
PFC
E
10
I EI UA Total
8 0 35 53
12 5 0 5 22
8 10 2 26 46
7 11 0 18 36
13 2 1 2 18
Abbreviations: E: excitation only; I: inhibition only; EI: excitation followed by inhibition; UA: unaffected.
BARRAGAN
AND FERREYRA-MOYANO
TABLE 3 NEURONS RECORDED IN HP Otthodromic Stimulation Site
Antidromic
E
I
EI
Unaffected
Total
AON MOB PFC VSPR Total
14 4 0 63 81
6 1 0 8 15
8 0 1 10 19
I 1 0 2 4
31 50 3 26 110
60 56 4 109 229
Abbreviations: E: excitation followed by inhibition.
only; I: inhibition only; EI: excitation
DISCUSSION
FIG. 2. Temporary cessation of spontaneous discharges in neurons recorded in BLA (A), AON (B), CA1 (C), and subiculum (D). following stimulation of VSPR (upper trace) or AON (lower trace) (A), of MOB (upper trace) or VSPR (lower trace) (B), of AON (upper trace) or VSPR (lower trace) (C). and of AON (upper trace) or VSPR (lower trace) (D). (A,D) Ten superimposed sweeps. (C,B) Five superimposed sweeps. Arrowheads indicate stimulus artifact. Calibration marks: Horizontal, 200 ms (A) and (B); 400 ms (C), and 100 ms (D). Vertical, 0.2 mV (A) and (B); 0.4 mV (C, upper trace); 1 mV (C, lower trace); 0.1 mV (D).
VSPR and the other to the AON (neurons in CA1 and subiculum), to the agranular insular ventral region in the PFC (cell bodies in BLA) and to the ipsilateral MOB (somas in the AON). All these neurons satisfied the requirements for branching specified in the Method section. Figure 3 illustrates four of these cells.
Our results demonstrate that several forebrain regions that include both cortical as well as quasi-cortical structures, such as the AON [25,42,53], MOB [46,48], and BLA [1,7] project axons to the region of the VSPR in the rat and receive, in turn, excitatory and/or inhibitory inputs from the later. The present study is the first to offer electrophysiological evidence for ipsilateral axonal branching of single AON neurons with collaterals innervating the medial ventral NA and the MOB. Our work also describes the functional characteristics of branched axonal neurons in the AON, which innervates both the NA and the OT. These neurons have been shown to be aspartatergic as well as gabaergic [8]. In our study, electrical stimulation of the AON and MOB antidromically activated many neurons in CA1 and subiculum (Table 3). In a recent study (54), neuroanatomical evidence has been provided for projections from the temporal one-third of CA1 and from the subiculum to the AON and MOB, among other forebrain regions. This connection might serve as a direct pathway for the retrograde transport of putative neurotoxins and/or viruses (which may use the olfactory receptor cells as a portal of entry to the CNS) directly to archicortical structures [ 141. According to Yim and Mogenson [57], stimulation of the BLA in the rat, orthodromically discharged neurons in VP regions with a mean latency of 11 ms. This is consistent with the mean latency of antidromically invaded neurons in BLA following VSPR stimulation in our cases, 12.3 ms., indicating that presumably, afferent fibers from BLA make a monosyn-
Variations in CV and Threshold Following Single Shock Conditioning Volleys to the VSPR TABLE 4 Thirty-five neurons in AON (n = 6), BLA (n = IO), HP (n = 5), and PFC (n = 14) antidromically driven following VSPR stimulation were tested for variations in CV and threshold using paired-pulse stimulation. Interstimulus intervals were varied between 1 and 1000 ms. Aftereffects were present only when conditioning stimuli were delivered at suprathreshold values for spike generation. Following the relative refractory period, an early supernormal period characterized by an increase in CV and a decrease in threshold was found in 9 (26%) neurons. The magnitude of the latency decrease ranged between 0.2 and 1.3 ms and the duration of the supernormal period between 15 and 200 ms. These effects were dependent on the presence of an AD spike in the conditioning response. Peak magnitude occurred at C/T intervals of lo-75 ms. Figure 4 illustrates one neuron that exhibited an increase in CV following a conditioning pulse (B) and another that did not showed significant variations in antidromic latency to a preceding stimulus (A).
EVOKED RESPONSES OF VSPR UNITS FOLLOWING AON. HP, MOB, AND PFC STIMULATION* Otthodromic Stimulation Site
Antidromic
E
I
EI
Unaffected
Total
AON HP MOB PFC Total
5 0 29 3 37
19 9 88 3 119
4 1 14 2 21
4 2 5 0 11
12 17 135 4 168
44 29 271 12 356
Abbreviations: E: excitation only; 1: inhibition only; EI: excitation followed by inhibition. * We have omitted figures from BLA stimulation because. for technical reasons, precise location of responsive units in VSPR was considered unreliable.
BASAL FOREBRAIN
333
INTERCONNECTIONS
PIG. 3. Examples of branched axon neurons. Neurons recorded in BLA (A and B). CA1 (C), and ventral subiculum (D). (A) Upper trace, antidromicallydischarged neuronfollowing agranular insular ventral stimulation. Lower trace shows collision-extinction (dot) of the antidromic spike when preceded by a conditioning stimulus to the VSPR which elicited an antidromic spike. (B) Shows that reversing the stimuli order, collision extinction (dot) follows the VSPR stimulus. (C) An antidromically invaded neuron following a VSPR stimulus (upper trace) is blocked (dot; middle trace) when an antidromic spike occurs within the collision interval following an AON stimulus. The lower trace shows that collision will not take place when the interval between the conditioning and test pulses is increased beyond the reciprocal collision time. In D (upper trace), VSPR stimulation elicits an antidromic spike in ventral subiculum. Middle trace shows collision-extinction (dot) of the antidromic spike, when preceded by a stimulus to AON which antidromically discharges the same neuron. Lower trace: collision fails to occur when the intensity of the conditioning pulse is subthresholdfor spike activation. Asterisks indicate antidromically discharged neurons. Dots, collision-extinction of the antidromic spike, and arrowheads, the stimulus artifact. Calibration marks: horizontal, IO ms (A, B, C, and D); vertical, 0.04 mV (A, B, and C); 0.1 mV (D).
aptic excitatory connection with neurons in ventrostriopallidal regions [57]. In addition, retrograde and anterograde tracing techniques have disclosed afferent projections from the BLA to the NA [26,28,35,36,40] and to the OT and medial and ventral parts of the caudate-putamen [27]. Some of these projections may utilize excitatory amino acids as a transmitter [8,44]. Electrophysiological studies suggest also an excitatory nature of the amygdaloidstriatal projection in the rat [11,57] and cat [37]. Recent studies have shown that in the rat, the amygdala has strong projections to most areas of the striatum, including the NA, OT, and parts of the caudatoputamen [26,28,45], that the main source of amygdalostriatal projection is the basolateral nucleus [28,45], that they are of an excitatory nature [ 11,37,57], and that these projections are topographically organized [26,28,45]. It has been suggested, on the basis of electrophysio-
logical findings, that the NA provides a link between the amygdala and the VP, and that this pathway may constitute a bridge between the limbic and motor systems [57]. Amygdalostriatal projections have been thoroughly described by de Olmos [12]. They course mainly in the ventral amygdalofugal pathway and stria terminalis. The amygdaloid input to the striatum originates primarily in the basolateral nucleus, although there is evidence for additional but lesser contributions from the basomedial nucleus and the amygdalo-hippocampal area. Except for the nonlimbic rostral part of the striatum and the adjacent divisions of the OT, the rest of the striatum is supplied by the ventral amygdalofugal system, which carries the bulk of the striatopetal amygdaloid projections. Retrograde tracing studies in the rat indicate the existence of a triangular relationship involving the BLA, PFC, and striatum, that is, specific amygdaloid domains project to particular cortical
334
BARRAGAN
AND FERREYRA-MOYANO
FIG. 4. Activity-dependent variations in conduction velocity. Examples of antidromically activated neurons in dorsal subiculum (A) and PFC (B) following stimulation of the VSPR. Control, unconditioned responses are shown in top traces. Middle and bottom traces illustrate responses when preceded by a conditioning pulse at IO and I5 ms, respectively. A significant reduction in antidromic latency (increased conduction velocity) was only observed in the PFC neuron. Asterisks indicate antidromic spikes. Three and four superimposed sweeps. Calibration marks: horizontal, 1 ms (A and B); vertical, 0. I mV (A) and 0.04 mV (B).
areas as well as to the principal
striatal targets of these same areas
[32]. For example, many amygdaloid neurons were double labeled when injections of different color dyes were made into the PFC and the striatal target of that same PFC subfield [31]. In our study, axonal branching in five BLA neurons was demonstrated with the use of the reciprocal collision test, with one branch directed to the lateral PFC in the region of the agranular insular dorsal zone and the other to the VSPR (see the Results section). These findings, therefore, strongly support the conclusions of MC Donald [3 11, because in all cases axonal stimulation in agranular insular dorsal cortex and in the VSPR satisfied the conditions for activation of different branches of the same axon. In our studies, we recorded antidromically activated units in the agranular insular ventral, lateral orbital, and ventrolateral orbital cortex (Tables 1 and 2) as well as in prefrontal region 2 (which is supposed to be a motor region) following stimulation of the VSPR zone, thus confirming the projections from prefrontal cortical areas to the VSPR [ 191. A corollary to these findings is that the organization in parallel circuits of different subdivisions of the PFC and a number of subcortical structures cannot be considered as isolated features, but that they must be regarded as components or aspects of the integrative functions of a circuit or network [19]. In addition, the ventrolateral orbital and lateral orbital regions have been shown by our group to be reciprocally linked to the ipsilateral MOB and contralateral MOB with both neuroanatomical [ 161 and electrophysiological techniques in the rat [lo] and armadillo [ 161. The polymorphic layer cells of the OT are noteworthy, because they largely project GABAergic terminals to the mediodorsal nucleus [29] (a thalamic structure involved in olfactory discrimination tasks), as well as being the origin of afferents to the nuclei gemini in the posterior lateral hypothalamus; therefore, the deep cells in the OT not only project to regions involved in processing of olfactory information but receive, in turn, afferents from the AON and, via an intermediate relay, from the PFC and the MOB itself. In addition, it has been demonstrated in physiological studies that olfactory-related information is being relayed to the geminiprojecting cells in the ventrostriopallidal system [47]. Therefore,
the available evidence tends to suggest that cells in the VSPR are furnished with inputs from several olfactory structures, such as the MOB, piriform cortex [4], and AON (this article), and that these VSPR neurons are in a position to relay olfactory-related information to other brain regions downstream in the olfactory pathway such as the mediodorsal thalamic nucleus [29] and nuclei gemini [24]. Our results demonstrate that conduction properties of some axons afferent to the VSPR are not invariant and that aftereffects of impulse activity can be manifested as variations in conduction velocity and excitability. In the present experiments, a supemorma1 period of increased conduction velocity, which resulted in a decrease in antidromic latency and an increase in excitability, was demonstrated in 25% of all neurons tested. The time course and magnitude of the decreases in antidromic latency were similar to those of a threshold decrease at the stimulation site, a fact that suggests that a depolarization of the axon follows its activation. A corollary to this finding is that, because conduction velocity varies with the history of impulse activity along the axon, a constant latency does not constitute a prerequisite for the identification of antidromically activated cells, nor does a variable latency constitute a sufficient condition for categorizing a unit as orthodromically activated. The phenomenon of activity-dependent latency variability may serve to modulate the postsynaptic efficiency of impulses in unmyelinated fibers, because the temporal summation of the latter spikes in a train would be reduced, as well as the transmitter quantum released by each impulse (due to tonic depolarization), therefore decreasing their effects on the postsynaptic membrane [3]. The possibility that antidromic invasion of nonspontaneously active neurons were due to direct current spread from the VSPR stimulating electrode to axons in regions surrounding the VSPR was evaluated and considered unlikely for the following reasons: a) constant current rather than voltage was routinely employed, allowing to accurately assess the antidromic threshold that fluetuated between 120 and 500 PA for axons with CV of 0.3 to 0.9 m/s. b) Inspection of a log-log current distance plot [43] indicates that a 200 ps cathodal monopolar pulse delivering a current
BASAL FOREBRAIN
INTERCONNECTIONS
of 400 PA, would only stimulate myelinated fibers (CV = 10 m/
s) confined within a radius of 1000 pm and cell bodies lying at a distance of 650 pm. It should be reminded that these values are for monopolar stimulation (we used bipolar stimulation) and for medium sized fibers. Much higher current values would be needed to stimulate fibers with CV of OS- 1.Om/s lying beyond the boundaries of the VSPR, because it is known that both in the peripheral, as well as in the CNS, the greater the CV of an axon, the less current it takes to stimulate it [6]. c) In two experiments, monopolar axonal microstimulation [2] was performed. Construction of depth-intensity curves revealed that in eight AON antidromically invaded neurons following stimulation of the VSPR, the activation threshold was achieved within a lo-30 pm displacement range. Beyond a distance of lOO- 150 pm from the point of lowest threshold, the latter increased progressively as the stimulating electrode was advanced or withdrawn; stimulation strengths up to 500 PA were unable to antidromically discharge the neurons, as the distance was increased to 300400 pm. ACKNOWLEDGEMENTS
This work was supported by grants from CONICET, CONICOR, FundaciQ Perez Companc, and Fundaci6n Antorchas.
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Pergamon 0361-9230(94)00281-9
Ventrostriopallidal Functional Interconnections With Cortical and Quasi-cortical Regions E. I. BARRAGAW AND H. FERREYRA-MOYANO*t’ */nstituto de lnvestigacidn Me’dica, Mercedes y Martin Ferreyra, Cdrdoba, Argentina tC&edra de Psicobiologh Experimental, Escuela de Psicologia-Facultad de Filosof,;3 y Humanidades, Universidad National de Cdrdoba, Cdrdoba, Argentina [Received 27 May 1994; Accepted 25 October 19941 its former boundaries: it is now clear that both the striatum and globus pallidus extend rostroventrally to reach the base of the brain, forming a ventral striatum (VS) and ventral pallidum (VP) [22]. On the basis of specific protein markers, DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein M 32.000) and synaptophysin, a marker for nerve terminals that also effectively labels pallidal synapses (according to Walaas and Ouimet [56]), the VP was traced rostroventrally from the globus pallidus to the superficial layers of the OT, moreover, the VS and the VP were found to be densely intermingled in parts of the OT and medial forebrain regions, and clearly separated in more caudal regions. These results indicate that the ventrostriopallidal region (VSPR), at least in the rat, extends both rostrocaudally and dorsoventrally, in a highly complex, intermingled fashion, throughout most of the basal forebrain [56]. On the other hand, the relationship of the VSPR with olfactory forebrain structures has not been clearly defined. For example, several authors have emphasized its close relationship to the olfactory system proper on the basis that a) some of its components, such as the OT, receive a direct input from the main olfactory bulb (MOB); b) part of the OT, especially its lateral region, fulfills the requirements for being classified as a cortex and, thus, can be considered to be part of the olfactory cortex [33,49]; and c) the entire rostrocaudal extent of the later is mapped onto the OT from lateral to medial [30]. Nevertheless, several investigators have emphasized the fact that this region, in spite of its layered configuration, should be considered as a ventral extension of the basal ganglia [21,23,34,39]. Others, however, seem to view the OT as a transition area between the olfactory cortex and the striatum [33,55]. An alternative way to clarify this controversy would be to functionally analyze putative reciprocal connections with the VSPR from forebrain regions known to be morphologically as well as functionally related to the olfactory system. The present study addresses the organization of descending as well as ascending projections from cortical and quasi-cortical regions to the VSPR in the rat. All of these structures, in addition, have reciprocal mono- or disynaptic connections with the MOB: the anterior olfactory nucleus (AON) [20], the basolateral nucleus of the amygdala (BLA) [13], the OT [1,4], the lateral and ventrolateral orbital cortex in the prefrontal region [10,18,41,51,52],
ABSTRACT: A total of 287 neurons were antidromically driven in quasi-cortical regions, i.e., anterior olfactory nucleus (24%), basolateral amygdala (13%), main olfactory bulb (4%), prefrontal cortex 137%1.and In the hippocampal formation (22%) following macro-’ and’&rostimula& of t6e rat’s ventrostriopallklal & glon (VSPR). In addiion, a substantial number of units @I= 175) were also transynaptiilly affected In all these structures by shocks delivered to the VSPR. Excitatory effects were detected in 50 neurons (56.1% of ntsponslve Cells), 36 cells (40.4%) responding with inhibition of spontaneous discharges. Conversely, stimulation of cortical and quasi-cortical regions antldromlcally discharged (n = 37) or transynaptically affected (n = 151) units in the VSPR; 168 neurons were not responsive to VSPR stimulation. Axon collaterallxation (branching) of 14 neurons in anterior olfactory nucleus, basolateral amygdela, and hippocampal formation was revealed wlth the use of the reciprocal collision test. Conduction properties of 35 neurons, evaluated by paired-pulse stimulation, indicated that only 23% showed a significant increase in conduction velocity and a decrease In threshold during the supernormal phase. The present findings first, described s&g interconnections between the neocorteX and striatal structures, and second, that the VSPR as suggested by previous structural, hodological, and hlstochemlcal stud& seems to maintain a more close relationship with olfactory related structures than hlthetio suspected. KEY WORDS: Basal ganglia, Ventral pallidum and strfatum, Antidromlc unlt drlvlng, Transynaptic effects, Main olfactory bulb, Anterior olfactory nucleus, Basolateral amygdala, Prefmntal cortex, Hlppocampal fotmatlon.
INTRODUCTION The basal forebrain
is a complex structure, which includes, among other regions, the substantia innominata, the basal nucleus of Meynert, the nucleus accumbens (NA), the olfactory tubercle (OT) and cortex (cortex piriformis), and the amygdaloid nuclei. Clinical, physiological, and behavioral studies have stressed the crucial role played by the basal forebrain in behaviors ranging from basic drives and emotions to higher cognitive functions [ 11. One of the basal forebrain components, the striatopallidal system, has recently undergone a considerable reevaluation in relation to
’ Requests for reprints should be addressed to H. Ferreyra-Moyano, INIMEC, Casilla de Correo 389, 5000, C6rdoba. Argentina. 329
330
BARRAGAN AND FERREYRA-MOYANO
and the comu Ammonis area 1 (CAI) region and subiculum in the hippocarnpal complex (HP) [54]. Antidromic activation of neurons was used as a reliable method for the topographical localization of cell bodies, provided certain constraints were met, which are described in the Method section. In addition, effects of VSPR stimulation were assessed on unit activity recorded throughout the AON, MOB, prefrontal cortex (PFC), BLA, and HP. A preliminary study has been published [5]. ABBREVIATIONS AON: anterior olfactory nucleus; BLA: basolateral nucleus of the amygdala; CA1 : comu Ammonis area 1; HP: hippocampal formation; MOB: main olfactory bulb; NA: nucleus accumbens; OT: olfactory tubercle; PFC: prefrontal cortex; RCT: reciprocal collision time; VP: ventral pallidum; VS: ventral striatum; VSPR: ventrostriopallidal region. METHOD Sixty-five rats (Wistar modified strain), 200-300 g, were anesthetized with Urethane (1.2 g/kg, IP) and mounted in a stereotaxic instrument with the incisor bar 3.3 mm lower than the ear bars, according to the stereotaxic atlas of Paxinos and Watson [38]. Core temperature was monitored with a digital rectal thermometer and maintained between 37 and 38°C throughout the experimental session. Concentric bipolar stimulating electrodes (Rhodes Med. Instruments, Inc., Model NEX-100, shaft diameter 0.5 mm) were advanced from the brain surface and positioned in the VSPR. Constant current monophasic square wave pulses between 0.1 and 1.3 mA in amplitude and 200 ms in duration were delivered through these electrodes at a rate of 0.2 to 1 Hz. Extracellular anti- and/or orthodromic unit responses were recorded throughout the AON, the MOB, the BLA, the hippocampus, and the PFC following VSPR stimulation. In a number of experiments, single-unit activity was recorded from the VSPR and responses to MOB, AON, BLA, and CA1 region and ventral subiculum stimulation was also evaluated. Extracellular recording of single unit activity and microstimulation was performed with stainless steel microelectrodes (A-M Systems, Inc., 12 Mfl impedance at 1000 Hz), which were advanced from the brain surface by means of an hydraulic microdrive (Narishige MO-8). Single-unit activity was amplified and displayed on a storage oscilloscope as filtered (300- 10 kHz bandpass) signals and monitored with a loudspeaker. Criteria for antidromic unit driving has been extensively discussed in previous articles [l&17]. Briefly, the evoked action potential was considered to arise from a single cell when: a) it had an all-or-none amplitude at threshold levels of stimulation; b) the evoked and spontaneously generated potentials had similar waveforms; c) the spike was able to consistently follow twin pulse stimulation at frequencies of 200 Hz or greater; d) in some cells, the waveform exhibited an initial segment and a somatodendritic component in the second of two closely spaced action potentials; and e) the antidromic spikes collided and were blocked by a spontaneous discharge at a critical time interval equal to twice the antidromic latency plus the absolute refractory period of the axon at the stimulated site. When a neuron was antidromically activated from two distinct sites of stimulation, this could have been due either to stimulation of the same axon at different sites or to stimulation of two collaterals of the same axon. The reciprocal collision test was used to distinguish between these two possibilities. The reciprocal collision time (RCT) corresponds to the maximal interval between the stimuli applied at both sites producing a blockade of the second antidromic spike.
When this RCT was greater than the difference between the latencies of the two antidromic responses plus the refractory period of the second stimulated axonal branch, it was concluded that the neuron was branched. Antidromic latency was measured from stimulus artifact to the base of the spike (first departure from baseline). Initial segment spikes followed by somato-dendritic components were observed in 23% of antidromically invaded cells. When only one component was present, it was assumed to be the somato-dendritic based on the total spike duration and the ratio of positive to negative phase duration. The modal value of ten antidromic latency measurements was assigned as the control latency for a given neuron to eliminate antidromic latency jitter that occurs during periods of spontaneous impulse activity [ 15,501. The estimated conduction distance between recording and stimulating electrodes was calculated by measuring the distance between the shafts of the stimulating and recording electrodes in the in situ preparation. This figure was later adjusted by estimating the distance between the blue spots (Prussian blue reaction) in coronal sections serially cut at 80 pm intervals. To compensate for spread of stimulus current, 0.2 mm was subtracted and this value multiplied by 1.2 to correct for possible nonlinear axonal trajectory, that is, a 20% increase in the distance separating the stimulating and recording sites was allowed to compensate for tortuosity in the axon’s pathway. In calculating estimated axon conduction velocity (CV), conduction time was obtained by subtracting 0.2 ms from the antidromic latency to compensate for the utilization time at the site of stimulation and delay of invasion near the cell body. Neuron excitability was assessed with the help of a threshold hunter. This device was designed and constructed in our laboratory [9], with the purpose of evaluating threshold variations in antidromically activated neurons before, during, and following the supernormal period of increased CV and decreased excitability. The technique is based in the method of constant response [2]: during threshold evaluation, the duration of an electric pulse applied to the neuron’s axon is automatically varied, depending on the presence or not of an action potential. For a given spike, the stimulus amplitude is progressively decremented (- delta i) up to a point where the neuron ceases to respond, and thereafter, the stimulus amplitude is progressively increased (+ delta i) until slightly suprathreshold values are obtained. The procedure guaranties a discharge probability of the neuron equivalent to 50% of all applied stimuli, and the simple monitoring of the stimulus amplitude is enough to obtain the threshold value for a predetermined intensity. For more details, refer to Cinelli and FerreyraMoyano [9]. At the end of the experiment, positive current (lo-20 mA) was passed through the recording and stimulating electrodes, and tip position was later identified as blue spots in frontal-cut sections treated with Potassium Ferrocianide and HCl (l%.)(prUsSian Blue reaction). The tip of the VSPR stimulating electrode was found to lie between 11.2 and 8.6 mm rostral to the interaural plane. The figures of Young et al. (58) were used to precisely match the blue spot in relation to the VS and VP stimulated areas. These included, in addition to the above-mentioned structures, the deep region of the OT, the boundary zone between the VP and NA, the VP and substantia innominata, and the VP and fundus striati. RESULTS Electrical stimulation of the VSPR resulted in antidromic driving of neurons in the AON, the BLA, MOB, PFC, and hip-
BASAL FOREBRAIN INTERCONNECTIONS TABLE 1 ANTIDROMICALLY DRIVEN UNITS FOLLOWING VSPR STIMULATION (n: = 287)
Smtctun
n
Percent of Total
AON BLA HP MOB PFC*
70 37 63 11 106
24 13 22 4 37
LatetlCy Mean+SD
cv m/s Mean 2 SD
8.9 + 13.1 2 14 2 8.3 + 11.7 2
0.6 0.4 0.7 0.9 0.3
4.12 6.25 5.07 3.23 4.28
? 2 + 2 k
0.33 0.16 0.27 0.54 0.18
CV: conduction velocity (m/s). HP: includes cells in CA1 and subiculum. SD: standard deviation. n = number of neurons tested. * Prefrontal region 1 (Frl), n = 4, Prefrontal region 2 (FR), n = 64; Lateral orbital region (LO), n = 15; Ventrolateral orbital region (VLO), n = 23.
FlG. I. A, B, C, D, E, F, G, and H show 23 dorsoventral penetrations of the recording microelectrodes at five different A-P planes [38]. Antidromically invaded cells following electrical stimulation of the VSPR are shown as small horizontal lines. Abbreviatiolrs:amg = basolateral amygdala; aon = anterior olfactory nucleus; cal = comu Ammonis area 1 (hippocampus); ent = entorhinal cortex; s = subiculum; fr = prefrontal cortex.
pocampal structures (CA1 and subiculum). Figure 1 shows 23 of 132 dorso-ventral penetrations of the recording microelectrode made at different antero-posterior forebrain planes; small horizontal lines in each tract indicate location of neurons that responded with an invariant latency and a discrete threshold to VSPR stimulation. Neurons antidromically driven in the MOB were found to lie at the level of the mitral cell layer. Table 1 shows the distribution of these neurons in the prosencephalon as well as some of their electrophysiological characteristics. Estimated CV for all these neurons indicate that they belong to the C-fiber type (CV < 1.3 m/s). Roughly 50% (n = 89) of spontaneously discharging neurons recorded in the AON, BLA, MOB, PFC, and HP were responsive to VSPR stimulation; excitation was present in 56% of responsive units, temporary inhibition of spontaneous discharges was observed in 40% and 4% of cells exhibited an initial period of excitation followed by inhibition (Table 2). A substantial number (45%) of those neurons in AON, BLA, HP, and MOB, which were transynaptically affected following
VSPR stimulation, showed inhibitory effects (Table 2). Inhibition, that is, temporary cessation of spontaneous discharges, was present in either antidromically driven cells when challenged with a conditioning volley to the VSPR or in spontaneously active cells (Fig. 2). Strikingly similar periods of cessation of ongoing spontaneous discharges were seen in units recorded in the AON, CA1 region, subiculum, and amygdala when stimulation was attempted from two different brain regions (Fig. 2). A total of 229 neurons were studied in the HP (CA1 and subiculum). Table 3 summarizes the effects of AON, MOB, PFC, and VSPR stimulation on these neurons. Eighty-one neurons were antidromically discharged following electrical stimulation of these regions; in addition, a large number of spontaneously discharging neurons in the HP exhibited a temporary cessation of spontaneous discharges following AON or VSPR stimulation. In a different experimental setup, effects of electrical stimulation of several prosencephalic structures (AON, MOB, PFC, and HP) were assessed on 356 neurons recorded in the VSPR. Table 4 shows that approximately 53% of neurons were either antidromically driven (n = 37) or were otherwise affected in their spontaneous
discharge
pattern (n = 151).
Branched Axon Neurons Fourteen neurons were identified in the AON (n = 5), BLA (n = 2), CA1 region (n = 3), and ventral subiculum (n = 4) that possessed bifurcated axons, with one branch directed to the TABLE 2 EVOKED RESPONSESOF
AON, BLA, HP, AND PFC UNITS FOLLOWING VSPR STIMULATION (n = 175)
Pattern of Response
AON
BLA
HP
MOB
PFC
E
10
I EI UA Total
8 0 35 53
12 5 0 5 22
8 10 2 26 46
7 11 0 18 36
13 2 1 2 18
Abbreviations: E: excitation only; I: inhibition only; EI: excitation followed by inhibition; UA: unaffected.
BARRAGAN
AND FERREYRA-MOYANO
TABLE 3 NEURONS RECORDED IN HP Otthodromic Stimulation Site
Antidromic
E
I
EI
Unaffected
Total
AON MOB PFC VSPR Total
14 4 0 63 81
6 1 0 8 15
8 0 1 10 19
I 1 0 2 4
31 50 3 26 110
60 56 4 109 229
Abbreviations: E: excitation followed by inhibition.
only; I: inhibition only; EI: excitation
DISCUSSION
FIG. 2. Temporary cessation of spontaneous discharges in neurons recorded in BLA (A), AON (B), CA1 (C), and subiculum (D). following stimulation of VSPR (upper trace) or AON (lower trace) (A), of MOB (upper trace) or VSPR (lower trace) (B), of AON (upper trace) or VSPR (lower trace) (C). and of AON (upper trace) or VSPR (lower trace) (D). (A,D) Ten superimposed sweeps. (C,B) Five superimposed sweeps. Arrowheads indicate stimulus artifact. Calibration marks: Horizontal, 200 ms (A) and (B); 400 ms (C), and 100 ms (D). Vertical, 0.2 mV (A) and (B); 0.4 mV (C, upper trace); 1 mV (C, lower trace); 0.1 mV (D).
VSPR and the other to the AON (neurons in CA1 and subiculum), to the agranular insular ventral region in the PFC (cell bodies in BLA) and to the ipsilateral MOB (somas in the AON). All these neurons satisfied the requirements for branching specified in the Method section. Figure 3 illustrates four of these cells.
Our results demonstrate that several forebrain regions that include both cortical as well as quasi-cortical structures, such as the AON [25,42,53], MOB [46,48], and BLA [1,7] project axons to the region of the VSPR in the rat and receive, in turn, excitatory and/or inhibitory inputs from the later. The present study is the first to offer electrophysiological evidence for ipsilateral axonal branching of single AON neurons with collaterals innervating the medial ventral NA and the MOB. Our work also describes the functional characteristics of branched axonal neurons in the AON, which innervates both the NA and the OT. These neurons have been shown to be aspartatergic as well as gabaergic [8]. In our study, electrical stimulation of the AON and MOB antidromically activated many neurons in CA1 and subiculum (Table 3). In a recent study (54), neuroanatomical evidence has been provided for projections from the temporal one-third of CA1 and from the subiculum to the AON and MOB, among other forebrain regions. This connection might serve as a direct pathway for the retrograde transport of putative neurotoxins and/or viruses (which may use the olfactory receptor cells as a portal of entry to the CNS) directly to archicortical structures [ 141. According to Yim and Mogenson [57], stimulation of the BLA in the rat, orthodromically discharged neurons in VP regions with a mean latency of 11 ms. This is consistent with the mean latency of antidromically invaded neurons in BLA following VSPR stimulation in our cases, 12.3 ms., indicating that presumably, afferent fibers from BLA make a monosyn-
Variations in CV and Threshold Following Single Shock Conditioning Volleys to the VSPR TABLE 4 Thirty-five neurons in AON (n = 6), BLA (n = IO), HP (n = 5), and PFC (n = 14) antidromically driven following VSPR stimulation were tested for variations in CV and threshold using paired-pulse stimulation. Interstimulus intervals were varied between 1 and 1000 ms. Aftereffects were present only when conditioning stimuli were delivered at suprathreshold values for spike generation. Following the relative refractory period, an early supernormal period characterized by an increase in CV and a decrease in threshold was found in 9 (26%) neurons. The magnitude of the latency decrease ranged between 0.2 and 1.3 ms and the duration of the supernormal period between 15 and 200 ms. These effects were dependent on the presence of an AD spike in the conditioning response. Peak magnitude occurred at C/T intervals of lo-75 ms. Figure 4 illustrates one neuron that exhibited an increase in CV following a conditioning pulse (B) and another that did not showed significant variations in antidromic latency to a preceding stimulus (A).
EVOKED RESPONSES OF VSPR UNITS FOLLOWING AON. HP, MOB, AND PFC STIMULATION* Otthodromic Stimulation Site
Antidromic
E
I
EI
Unaffected
Total
AON HP MOB PFC Total
5 0 29 3 37
19 9 88 3 119
4 1 14 2 21
4 2 5 0 11
12 17 135 4 168
44 29 271 12 356
Abbreviations: E: excitation only; 1: inhibition only; EI: excitation followed by inhibition. * We have omitted figures from BLA stimulation because. for technical reasons, precise location of responsive units in VSPR was considered unreliable.
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333
INTERCONNECTIONS
PIG. 3. Examples of branched axon neurons. Neurons recorded in BLA (A and B). CA1 (C), and ventral subiculum (D). (A) Upper trace, antidromicallydischarged neuronfollowing agranular insular ventral stimulation. Lower trace shows collision-extinction (dot) of the antidromic spike when preceded by a conditioning stimulus to the VSPR which elicited an antidromic spike. (B) Shows that reversing the stimuli order, collision extinction (dot) follows the VSPR stimulus. (C) An antidromically invaded neuron following a VSPR stimulus (upper trace) is blocked (dot; middle trace) when an antidromic spike occurs within the collision interval following an AON stimulus. The lower trace shows that collision will not take place when the interval between the conditioning and test pulses is increased beyond the reciprocal collision time. In D (upper trace), VSPR stimulation elicits an antidromic spike in ventral subiculum. Middle trace shows collision-extinction (dot) of the antidromic spike, when preceded by a stimulus to AON which antidromically discharges the same neuron. Lower trace: collision fails to occur when the intensity of the conditioning pulse is subthresholdfor spike activation. Asterisks indicate antidromically discharged neurons. Dots, collision-extinction of the antidromic spike, and arrowheads, the stimulus artifact. Calibration marks: horizontal, IO ms (A, B, C, and D); vertical, 0.04 mV (A, B, and C); 0.1 mV (D).
aptic excitatory connection with neurons in ventrostriopallidal regions [57]. In addition, retrograde and anterograde tracing techniques have disclosed afferent projections from the BLA to the NA [26,28,35,36,40] and to the OT and medial and ventral parts of the caudate-putamen [27]. Some of these projections may utilize excitatory amino acids as a transmitter [8,44]. Electrophysiological studies suggest also an excitatory nature of the amygdaloidstriatal projection in the rat [11,57] and cat [37]. Recent studies have shown that in the rat, the amygdala has strong projections to most areas of the striatum, including the NA, OT, and parts of the caudatoputamen [26,28,45], that the main source of amygdalostriatal projection is the basolateral nucleus [28,45], that they are of an excitatory nature [ 11,37,57], and that these projections are topographically organized [26,28,45]. It has been suggested, on the basis of electrophysio-
logical findings, that the NA provides a link between the amygdala and the VP, and that this pathway may constitute a bridge between the limbic and motor systems [57]. Amygdalostriatal projections have been thoroughly described by de Olmos [12]. They course mainly in the ventral amygdalofugal pathway and stria terminalis. The amygdaloid input to the striatum originates primarily in the basolateral nucleus, although there is evidence for additional but lesser contributions from the basomedial nucleus and the amygdalo-hippocampal area. Except for the nonlimbic rostral part of the striatum and the adjacent divisions of the OT, the rest of the striatum is supplied by the ventral amygdalofugal system, which carries the bulk of the striatopetal amygdaloid projections. Retrograde tracing studies in the rat indicate the existence of a triangular relationship involving the BLA, PFC, and striatum, that is, specific amygdaloid domains project to particular cortical
334
BARRAGAN
AND FERREYRA-MOYANO
FIG. 4. Activity-dependent variations in conduction velocity. Examples of antidromically activated neurons in dorsal subiculum (A) and PFC (B) following stimulation of the VSPR. Control, unconditioned responses are shown in top traces. Middle and bottom traces illustrate responses when preceded by a conditioning pulse at IO and I5 ms, respectively. A significant reduction in antidromic latency (increased conduction velocity) was only observed in the PFC neuron. Asterisks indicate antidromic spikes. Three and four superimposed sweeps. Calibration marks: horizontal, 1 ms (A and B); vertical, 0. I mV (A) and 0.04 mV (B).
areas as well as to the principal
striatal targets of these same areas
[32]. For example, many amygdaloid neurons were double labeled when injections of different color dyes were made into the PFC and the striatal target of that same PFC subfield [31]. In our study, axonal branching in five BLA neurons was demonstrated with the use of the reciprocal collision test, with one branch directed to the lateral PFC in the region of the agranular insular dorsal zone and the other to the VSPR (see the Results section). These findings, therefore, strongly support the conclusions of MC Donald [3 11, because in all cases axonal stimulation in agranular insular dorsal cortex and in the VSPR satisfied the conditions for activation of different branches of the same axon. In our studies, we recorded antidromically activated units in the agranular insular ventral, lateral orbital, and ventrolateral orbital cortex (Tables 1 and 2) as well as in prefrontal region 2 (which is supposed to be a motor region) following stimulation of the VSPR zone, thus confirming the projections from prefrontal cortical areas to the VSPR [ 191. A corollary to these findings is that the organization in parallel circuits of different subdivisions of the PFC and a number of subcortical structures cannot be considered as isolated features, but that they must be regarded as components or aspects of the integrative functions of a circuit or network [19]. In addition, the ventrolateral orbital and lateral orbital regions have been shown by our group to be reciprocally linked to the ipsilateral MOB and contralateral MOB with both neuroanatomical [ 161 and electrophysiological techniques in the rat [lo] and armadillo [ 161. The polymorphic layer cells of the OT are noteworthy, because they largely project GABAergic terminals to the mediodorsal nucleus [29] (a thalamic structure involved in olfactory discrimination tasks), as well as being the origin of afferents to the nuclei gemini in the posterior lateral hypothalamus; therefore, the deep cells in the OT not only project to regions involved in processing of olfactory information but receive, in turn, afferents from the AON and, via an intermediate relay, from the PFC and the MOB itself. In addition, it has been demonstrated in physiological studies that olfactory-related information is being relayed to the geminiprojecting cells in the ventrostriopallidal system [47]. Therefore,
the available evidence tends to suggest that cells in the VSPR are furnished with inputs from several olfactory structures, such as the MOB, piriform cortex [4], and AON (this article), and that these VSPR neurons are in a position to relay olfactory-related information to other brain regions downstream in the olfactory pathway such as the mediodorsal thalamic nucleus [29] and nuclei gemini [24]. Our results demonstrate that conduction properties of some axons afferent to the VSPR are not invariant and that aftereffects of impulse activity can be manifested as variations in conduction velocity and excitability. In the present experiments, a supemorma1 period of increased conduction velocity, which resulted in a decrease in antidromic latency and an increase in excitability, was demonstrated in 25% of all neurons tested. The time course and magnitude of the decreases in antidromic latency were similar to those of a threshold decrease at the stimulation site, a fact that suggests that a depolarization of the axon follows its activation. A corollary to this finding is that, because conduction velocity varies with the history of impulse activity along the axon, a constant latency does not constitute a prerequisite for the identification of antidromically activated cells, nor does a variable latency constitute a sufficient condition for categorizing a unit as orthodromically activated. The phenomenon of activity-dependent latency variability may serve to modulate the postsynaptic efficiency of impulses in unmyelinated fibers, because the temporal summation of the latter spikes in a train would be reduced, as well as the transmitter quantum released by each impulse (due to tonic depolarization), therefore decreasing their effects on the postsynaptic membrane [3]. The possibility that antidromic invasion of nonspontaneously active neurons were due to direct current spread from the VSPR stimulating electrode to axons in regions surrounding the VSPR was evaluated and considered unlikely for the following reasons: a) constant current rather than voltage was routinely employed, allowing to accurately assess the antidromic threshold that fluetuated between 120 and 500 PA for axons with CV of 0.3 to 0.9 m/s. b) Inspection of a log-log current distance plot [43] indicates that a 200 ps cathodal monopolar pulse delivering a current
BASAL FOREBRAIN
INTERCONNECTIONS
of 400 PA, would only stimulate myelinated fibers (CV = 10 m/
s) confined within a radius of 1000 pm and cell bodies lying at a distance of 650 pm. It should be reminded that these values are for monopolar stimulation (we used bipolar stimulation) and for medium sized fibers. Much higher current values would be needed to stimulate fibers with CV of OS- 1.Om/s lying beyond the boundaries of the VSPR, because it is known that both in the peripheral, as well as in the CNS, the greater the CV of an axon, the less current it takes to stimulate it [6]. c) In two experiments, monopolar axonal microstimulation [2] was performed. Construction of depth-intensity curves revealed that in eight AON antidromically invaded neurons following stimulation of the VSPR, the activation threshold was achieved within a lo-30 pm displacement range. Beyond a distance of lOO- 150 pm from the point of lowest threshold, the latter increased progressively as the stimulating electrode was advanced or withdrawn; stimulation strengths up to 500 PA were unable to antidromically discharge the neurons, as the distance was increased to 300400 pm. ACKNOWLEDGEMENTS
This work was supported by grants from CONICET, CONICOR, FundaciQ Perez Companc, and Fundaci6n Antorchas.
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