In one group of experiments (n=3) retrograde fluorescent tracers occupied restricted sites of the basal nuclei of the amygdala (, ). In a second group of experiments (n=4 hemispheres) the bidirectional tracer BDA occupied extensive parts of the basal complex of the amygdala (). In all cases, labeled projection neurons and axonal terminals were found in nearly all prefrontal areas, but varied in density in areas and distinct layers, as elaborated below.
Composite of the injection sites in the amygdala
Prefrontal projection neurons directed to the amygdala
Caudal medial and orbitofrontal areas issued the most robust projections to the amygdala (-), as summarized for pooled data and mapped in . The highest densities of projection neurons were noted in medial area 25 (M25), dorsal area 24 (D24), and the orbitofrontal area OPro, in spite of the fact that the injection sites were centered in different parts of the amygdala (; ; ; ). A cluster analysis based on the profile of projections resulting from different injection sites showed that cases with a predominant involvement of the medial nuclei clustered together, while those involving principally the basolateral (BL) nucleus formed another cluster (). This confirmed the consistency of labeling after injection of specific sectors of the amygdala.
Origin of projection neurons directed to the amygdala from the prefrontal cortices in three cases
Pattern of input and output connections linking the left prefrontal cortex with the left amygdala
Distribution and density of output projections from the prefrontal cortex to the amygdala
Pattern of input and output connections linking the right prefrontal cortex with the right amygdala
The specificity of projections to restricted sites of the amygdala was evident after an injection confined to the basomedial nucleus (BM, also known as accessory basal; ), which resulted in large numbers of projection neurons in the medial part of area 25 (M25, 53%). Area 24 included a substantial proportion of labeled neurons (33−36%) when the tracer injection included the ventrolateral part of BL and a small part of the adjacent part of the lateral (L) nucleus (, case BB_L; , case AW_L).
As in medial prefrontal areas, most projection neurons from orbitofrontal cortex were found in its posterior sector. Area OPro included the highest proportion of projection neurons directed to the amygdala (; -), especially when injections included the intermediate part of the basolateral (BLi) nucleus (20−23%). The adjacent orbital area OPAll, area 13, and orbital area 12 (O12) also included moderate numbers of projection neurons directed to the amygdala. Rostral orbitofrontal cortices, including orbital area 14 (O14), 11, and orbital area 25 (O25) issued lighter projections (-).
Overall, projections from lateral prefrontal cortices were significantly sparser than from medial and orbitofrontal areas, and most arose from the ventrolaterally situated area 12 (L12; -). Ventral area 46 (V46) also included a few labeled neurons in most cases, except one case where the injection was restricted to the BM nucleus (; case AX). Other lateral prefrontal areas included few, if any, labeled neurons, suggesting that lateral prefrontal projections to the amygdala originate primarily from its ventral sectors, and project preferentially to the basolateral nucleus of the amygdala. Area 10 stood apart from other areas with the sparsest projections to the amygdala emanating from either its medial or lateral sectors.
Caudal orbitofrontal and caudal medial prefrontal cortices differ in their laminar organization from rostral orbitofrontal, rostral medial, and lateral prefrontal areas, so we grouped data from different areas based on their cortical type into four categories, as follows: agranular cortices included those lacking layer 4 (areas MPAll and OPAll); dysgranular areas included areas with a poorly developed layer 4 (areas 24, 25, 32, 13, and OPro); eulaminate areas included those with six layers, which were divided into two groups: eulaminate I (areas 14, 11, 10, 12, and 9) and eulaminate II cortices (areas 8 and 46), based on the distinction of their 6 layers, which is higher in eulaminate II than in I (Dombrowski et al., 2001
). This analysis revealed significant differences in projection density among different types of prefrontal cortices (single-factor ANOVA, F (3,24) = 25.39, P<0.00001).
Laminar organization of prefrontal projections to the amygdala
Normalized data from each case were pooled and are shown in . Most labeled neurons were found in cortical layer 5. Projection neurons in layers 2 and 3 were found in significant numbers only in caudal medial (areas MPAll, 32, 25, 24) and caudal orbitofrontal areas (OPAll, and OPro). Posterior orbitofrontal areas (areas OPAll, OPro) were distinguished by a comparable distribution of projection neurons in the upper (2−3) and deep (5−6) layers, as were caudal medial areas (MPAll, V24; ). Nevertheless, projections from superficial layers did not exceed projections from the deep layers in any prefrontal area. There were only a few labeled neurons in layer 6, found mostly in caudal medial and orbitofrontal areas, or in areas V46 and L12.
Axonal terminations from the amygdala in prefrontal cortices
We next investigated the extent of labeled axonal terminations from the amygdala in prefrontal cortices in four hemispheres of two animals with injection of BDA (cases BB and BD; -). Prefrontal connections with the amygdala are ipsilateral, so terminations in each hemisphere are considered to be independent.
Axonal terminals from the amygdala were found in all areas and layers of the prefrontal cortex, but varied substantially in density across areas. The highest densities were found in caudal orbitofrontal and caudal medial prefrontal cortices (areas OPAll, OPro, M25, MPAll, and 24). In contrast, rostral orbitofrontal, rostral medial, and lateral prefrontal areas included considerably lower densities of boutons (, , ). Analysis of projection density of areas grouped into four categories according to cortical type (as described above) revealed that the density of axonal boutons from the amygdala differed significantly among different types of prefrontal cortices (single-factor ANOVA, F(3,19) = 7.81, P < 0.01). Caudal agranular and dysgranular cortices (found in the caudal orbitofrontal and medial prefrontal cortex) received the highest density of axonal terminals. In contrast, the density of terminations in eulaminate areas in rostral orbitofrontal, rostral medial and lateral prefrontal cortices was comparatively low.
Distribution and density of axonal terminals from the amygdala in prefrontal cortices
Laminar pattern of terminations from the amygdala in prefrontal cortices
Axonal boutons from the amygdala assumed several distinguishable patterns. The most prominent pattern consisted of terminations distributed in two bands parallel to the pial surface. One band innervated superficial layers 1, 2, or both, and the other the deep part of layer 5 and layer 6 (; red arrowheads). In another pattern, columns of axonal terminals innervated all cortical layers. In caudal medial and orbitofrontal areas, the columns were broad (>1mm in w idth; , green arrowheads), and small in anterior prefrontal areas (<1mm in width; , green arrowheads). Another pattern showed patches of axonal terminals clustered in the superficial (layers 1, 2; , yellow arrowheads), middle (layer 4 and surrounding parts of layers 3 and 5; , yellow arrowhead), or deep (layers 5 and 6; , yellow arrowheads) layers of the cortex. The patchy pattern of innervation was mostly seen in rostral prefrontal areas. In a few rostral areas (e.g., area O14), there was occasional unilaminar innervation of layer 1 (; blue arrowhead).
Patterns of axonal terminations from the amygdala in prefrontal cortices
We further investigated the laminar specificity of amygdalar innervation of prefrontal cortex using density data for individual layers of each area. shows the relative density of boutons across areas as well as their distribution within layers of each area. Layers 1 and 2 of most medial and orbitofrontal areas included the highest density of boutons. Caudal medial and orbitofrontal areas (areas MPAll, M25 and OPAll) had the highest density of labeled boutons in layer 1, while layer 2 of areas 24, OPro, L12, 32, 14, and medial area 9 (M9) was the most densely innervated (). Other prefrontal cortices included relatively balanced densities of boutons in their superficial and deep layers, suggesting a true bilaminar innervation by the amygdala in these areas. In general, layer 6 of most lateral prefrontal areas included the highest density of boutons, except area D9, where layer 5 had the highest density. In addition, layer 6 was the most densely innervated layer of orbitofrontal areas 13 and 11, and frontal polar area 10. Areas V24 and O25 showed a unique innervation of their middle layers, including layer 4. Areas OPAll, OPro, and M25 also had high densities of boutons in their middle layers, though they lack, or have a poorly developed, layer 4. Layer 3, in general, was sparsely innervated and no area of the prefrontal cortex included a predominant distribution of boutons in layer 3. However, in areas OPro, OPAll, M25 and to a lesser extent in area 24, significant densities of labeled boutons were noted in layer 3 ().
We then pooled laminar data to determine the relative density of boutons in superficial (1−3) and deep (4−6) layers across areas, as shown in . In most areas the percentage of axonal terminals in superficial layers exceeded the deep, particularly in agranular (MPAll, OPAll) and dysgranular (D24, M25 and OPro) cortices (). In other dysgranular and eulaminate cortices, the density of axonal terminals was nearly equal in the superficial and deep layers. The proportion of axonal terminals in the deep layers was slightly higher than the superficial in only a few areas, including orbitofrontal areas 11 and 12 ().
Comparison of the input and output zones of prefrontal cortices connected with the amygdala
We next compared the relative density of projection neurons to axonal terminals in prefrontal cortices connected with the amygdala. The goal was to determine the extent to which prefrontal areas were predominantly receivers of input from the amygdala, or senders of projections to the amygdala. This was accomplished using normalized data, by expressing the estimated number of boutons for each area as a percentage of the sum of boutons in all prefrontal areas (), and by applying an analogous normalization to the number of projection neurons found in prefrontal areas. In some prefrontal areas the percentage of input from the amygdala significantly exceeded the percentage of output from the same area to the amygdala (, green coded, I > O). Of the heavily innervated caudal medial prefrontal areas, this category included area MPAll (). The medial parts of areas 9 (M9) and 10 (M10) also belonged to the category I > O, as did lateral areas 8, dorsal area 46 (D46), and D9, but the density of amygdalar innervation was substantially lower. Caudal orbitofrontal areas OPAll, OPro, O25, and 13 also belonged to the category I > O, although the differences in percentages of input and output were not significant (). The second pattern included prefrontal cortices with significantly higher proportion of output compared to input (, red coded, O > I), and included caudal medial areas 24, M25, and 32, and all rostrally situated orbitofrontal areas (11, O14, O12, and 10). On the lateral surface, areas dorsal 10 (D10), L12, and V46 were in the category O > I. These findings are summarized in . takes into account the overall density of connections, showing prefrontal areas possessing particularly strong links with the amygdala towards the top of the diagram, and also indicates the input-output characteristics of areas. ‘Senders’ (projecting more strongly to, than receiving projections from, the amygdala) are on the left and ‘receivers’ (showing the opposite balance of projections) are shown on the right of the figure.
Relative proportion of input and output connections in prefrontal cortices linking them with the amygdala
We then investigated the input and output connections for the superficial and deep layers of prefrontal cortices and the results are summarized in . By analogy with sensory corticocortical connections, projection neurons from the superficial layers (2−3) in prefrontal cortices directed to the amygdala may be considered ‘feedforward’, and axonal terminations from the amygdala terminating in the upper layers (1-upper 3) of prefrontal cortices may be considered 'feedback'. Only a few prefrontal areas showed a balanced form of this pattern, and included caudal areas D24, M25, and OPro (). Interestingly, feedback input from the amygdala in the superficial layers was widespread and included most medial and orbitofrontal areas (). Medial area MPAll was distinguished for receiving substantial feedback input from the amygdala but not reciprocating with a significant output to the amygdala. Feedforward input from the amygdala to the middle layers of prefrontal cortex, and feedback output from the deep layers of prefrontal cortex was more widespread and included nearly all medial and orbitofrontal cortices. Areas that received a relatively high proportion of feedforward input from the amygdala into their middle layers included the caudally situated medial and orbitofrontal cortices (areas MPAll, M25, OPAll, OPro, and 13; ). Feedback output from the prefrontal cortices, however, was not as evenly distributed. Areas D24 and M25 included a significantly high percentage of feedback output among prefrontal areas (). Nearly all orbitofrontal areas as well as lateral area 12 provided substantial feedback projections to the amygdala. A population analysis of the relationship of ‘feedforward’ prefrontal projection neurons from layers 2−3 to ‘feedback’ terminations from the amygdala in prefrontal layers 1- upper 3 revealed a significant correlation (r=0.60, P=0.003; ). In , for example, the placement of area V24 indicates that 29% of its projection neurons directed to the amygdala (shown on x-axis) originated from layers 2 and 3, and the complementary 71% from layers 5 and 6 (not shown), while 57% of the amygdalar terminations in area V24 (shown on y-axis) were found in layers 1 through 3, and the remaining 43% in layers 4 through 6 (not shown).
Relationship of laminar-specific input to output connections linking prefrontal cortices with the amygdala
The relationship of amygdalar connections to neurochemical classes of inhibitory neurons in prefrontal cortices
An important component of cortical circuits is their relationship with GABAergic interneurons. We addressed this issue by determining the relationship of prefrontal connections with the amygdala to two neurochemical classes of local inhibitory neurons that are positive for the calcium binding proteins CB and PV. These neurochemical classes of inhibitory neurons have a distinct laminar distribution in prefrontal cortices (Gabbott and Bacon, 1996
; Dombrowski et al., 2001
). We conducted a quantitative analysis to determine the number of CB and PV interneurons within a 75 μm radius from labeled projection neurons in four cases with BDA injection in the amygdala (cases BBr; BDr; BBl; BDl; ). In medial areas D24, and M25, and orbitofrontal areas OPAll, and OPro more CB interneurons surrounded projection neurons directed to the amygdala than did PV interneurons. Combined, these prefrontal areas included the largest percentage (~ 50%) of projection neurons directed to the amygdala. Other medial and orbitofrontal areas included equal numbers of CB and PV interneurons associated with each projection neuron (areas V24, 13, and O25).
Examples of the relationship of prefrontal CB and PV interneurons to prefrontal connections with the amygdala are shown in . Caudal medial and orbitofrontal areas included higher associations with CB than PV interneurons (areas D24, M25, OPAll, and OPro). Areas V24 and L12, both biased ‘senders’ of projections, were unique among prefrontal areas by having on average more PV positive interneurons surrounding each projection neuron directed to the amygdala than other medial and orbitofrontal areas. These two areas together provided approximately 12% of projection neurons directed to the amygdala, substantially fewer than areas where projection neurons were strongly associated with CB interneurons. The few projection neurons found in lateral prefrontal cortices, other than area L12, were mostly surrounded by PV interneurons, and contributed about 6% of the projection neurons to the amygdala.
Prefrontal connections with the amygdala overlap with the neurochemical classes of calbindin (CB) and parvalbumin (PV) positive inhibitory neurons in prefrontal cortices
Axonal terminals from the amygdala overlapped largely with CB interneurons in layers 2 and upper 3, where CB interneurons predominate (). The axonal terminals from the amygdala in some prefrontal areas also targeted the PV-dominated middle layers, although their densities were substantially lower ().