Iontophoretic tracer delivery sites
Three rats (cases 09-133, 09-110, and 10-9) with dual iontophoretic delivery sites that were most accurately targeted and restricted to the CEAm and BSTvl were selected for quantitative analysis of retrograde labeling. In cases 09-110 and 09-133, FG was iontophoresed into the CEAm (see ) and CTB was iontophoresed into the BSTvl (see ). In case 10-9, the tracers delivered into each target were switched (see , ). Retrograde labeling patterns were consistent across these three cases, although the actual numbers of retrogradely labeled neurons varied (). Different patterns of retrograde labeling resulted from tracer delivery sites that “missed” the CEAm and BSTvl, and were instead centered in closely adjacent regions. Those findings are presented at the end of the results section.
Of the three selected cases with the most accurate tracer delivery sites, case 09-133 consistently had the largest number of retrogradely labeled neurons across quantified brain regions. This representative case was used to digitally plot the distribution of tracer-labeled neurons projecting to the CEAm and BSTvl (see –).
Fig. 3 Distribution maps of retrogradely labeled neurons within the caudal medulla. CEAm- and BSTvl-projecting neurons were located throughout the caudal NTS and VLM, but were most prevalent between bregma levels −14.86 and −13.60 (a–e (more ...)
Fig. 12 Distribution maps of retrogradely labeled neurons within the temporal cortex/posterior amygdala. large numbers of retrogradely labeled neurons were present within the BLAp and PA of the amygdala, TR of the cortex, and CA1 region of the ventral hippocampus. (more ...)
In general, the large majority of brain regions that contained CEAm-projecting neurons also contained BSTvl-projecting neurons, and vice versa. However, and as described in more detail in the following sections, some regions projected primarily to the CEAm with comparably less input to the BSTvl (e.g., insular cortex). Some regions displayed the opposite pattern, with relatively more input to the BSTvl compared to the CEAm (e.g., NTS), while some brain regions projected equivalently to both the CEAm and the BSTvl (e.g., PVT). Across all the regions in which retrograde labeling was quantified, double-labeled neurons with axonal projections to both the CEAm and the BSTvl accounted for as little as 2% to as much as 13% of the total population of tracer-labeled neurons (). The distribution of retrograde labeling is plotted in –, in which red stars represent individual CEAm-projecting neurons, and green circles represent BSTvl-projecting neurons.
Fig. 10 Rostrocaudal distribution of retrogradely labeled neurons within the PVT. a larger numbers of CEAm-projecting neurons (red) were located within the cPVT (−4.20 to −3.50), while larger numbers of BSTvl-projecting neurons (green) were located (more ...)
Retrogradely labeled neurons in the medulla were located primarily within the caudal, visceral portion of the NTS and the caudal VLM ipsilateral to the tracer delivery sites. Significantly more NTS neurons projected to the BSTvl compared to the number of NTS neurons projecting to the CEAm (136 ± 6 vs. 46 ± 15 neurons; P = 0.005). Rostrocaudal analysis of retrograde labeling patterns revealed a different distribution of BSTvl- versus CEAm-projecting NTS neurons (, ). Two-way ANOVA revealed a significant effect of both tracer target site [F(1, 29) = 90.39, P < 0.001] and rostrocaudal NTS level [F(4, 29) = 4.26, P < 0.05] on the number of retrogradely labeled NTS neurons, as well as a significant interaction between tracer target site and rostrocaudal level [F(4, 29) = 6.17, P < 0.01]. At levels caudal to the area postrema (AP), the NTS contained relatively few CEAm-projecting neurons (red stars in ). The number of CEAm-projecting NTS neurons increased at more rostral levels, reaching a peak just rostral to the AP (, ). Conversely, the number of BSTvl-projecting NTS neurons reached a peak at the mid-AP level (green circles, , ). NTS neurons with collateralized projections to both the CEAm and BSTvl were most prevalent in sections just rostral to the AP (). Approximately 9% of all tracer-labeled NTS neurons were double-labeled (). Overall, 33 ± 4% of all CEAm-projecting NTS neurons collateralized to also provide input to the BSTvl, whereas only 11 ± 4% of all BSTvl-projecting NTS neurons collateralized to innervate the CEAm ().
Fig. 4 Rostrocaudal distribution of retrogradely labeled NTS neurons. a The number of BSTvl-projecting neurons (green) peaked at the mid-AP section level (−14.16 mm from bregma), while smaller numbers of CEAm-projecting neurons (red) were distributed (more ...)
Retrogradely labeled VLM neurons were present at the same rostrocaudal levels as retrogradely labeled NTS neurons (). Quantification of labeling within the VLM revealed a nonsignificant trend towards a greater number of BSTvl- versus CEAm-projecting neurons (25 ±11 vs. 8 ± 4 neurons; P = 0.63). VLM neurons projecting to the CEAm or BSTvl were distributed uniformly across rostrocaudal levels, and moderate numbers of double-labeled neurons with collateralized projections to both the CEAm and BSTvl were observed (). Approximately 11% of all tracer-labeled VLM neurons were double-labeled, similar to proportions of double-labeled neurons within the NTS ().
Pontine input to the CEAm and BSTvl was predominantly confined to the parabrachial nucleus (PB; ). The locus coeruleus contained a small number of CEAm- and BSTvl-projecting neurons (i.e., 1–3 neurons per section, data not shown). Retrogradely labeled neurons were distributed bilaterally within several PB subnuclei, although a strong ipsilateral predominance of labeling was evident (). At the caudal end of the PB (), retrograde labeling within the PB was heavily concentrated within the ventral lateral (PBlv), medial (PBm), and waist subnuclei (PBw). Within these subnuclei, CEAm-projecting neurons significantly outnumbered BSTvl-projecting neurons (PBlv = 79 ± 9 vs. 29 ± 6 neurons; P = 0.01; PBm = 139 ± 25 vs. 45 ± 7 neurons; P = 0.02; PBw = 25 ± 4 vs. 11 ± 1 neurons; P = 0.04). Relatively few (i.e., approximately 6 ± 1, 3 ± 1, and 8 ± 2%) of all tracer-labeled neurons within the PBlv, PBm, and PBw were double-labeled (). More rostral levels of the PB () contained larger numbers of CEAm- and BSTvl-projecting neurons that were primarily localized within the external lateral PB subnucleus (PBle; see ), with fewer retrogradely labeled neurons present within the PBlv, PBm, and PBw. Quantification revealed a nonsignificant trend towards larger numbers of PBle neurons projecting to the CEAm versus the BSTvl (298 ± 72 vs. 130 ± 9 neurons; P = 0.08). In contrast to other PB subnuclei, many of the retrogradely labeled PBle neurons collateralized to innervate both the CEAm and BSTvl: approximately 12 ± 3% of all tracer-labeled PBle neurons were double-labeled (). When double-labeled neurons were excluded, significantly more single-labeled PBle neurons projected to the CEAm compared to the number that projected to the BSTvl (; P < 0.05).
Fig. 5 Distribution maps of retrogradely labeled pontine neurons, which were primarily located in the PB between bregma levels −9.80 and −9.25. Generally, CEAm-projecting PB neurons (red stars) were more prevalent than BSTvl-projecting PB neurons (more ...)
Fig. 13 Confocal z-stack images of selected brain regions containing large numbers of CEAm- and BSTvl-projecting neurons and a relatively high incidence of double-labeling: PBle (a, compare to ), BMA (d, compare to ), and BLAp (e, compare to (more ...)
Within the caudal midbrain (), CEAm- and BSTvl-projecting neurons were distributed ventral to the central aqueduct, within the pedunculopontine nucleus (PPN), dorsal raphé (DR), and ventral lateral periaqu-eductal gray (PAGvl). Within the DR, there was no significant difference between the numbers of CEAm- and BSTvl-projecting neurons (77 ± 20 vs. 65 ± 6 neurons; P = 0.61), and relatively few tracer-labeled DR neurons (7 ± 1 neurons, 6 ± 2% of total) had collateralized projections to both the CEAm and BSTvl (). In more rostral midbrain sections, retrograde labeling extended ventrally to include the central linear raphé (CLI; ). There was no significant difference between the number of CEAm- and BSTvl-projecting CLI neurons (29 ± 9 vs. 35 ± 6 neurons; P = 0.62), and there was very little collateralization of individual CLI neurons to both the CEAm and BSTvl ().
Fig. 6 Distribution maps of retrogradely labeled neurons within the caudal midbrain. Retrogradely labeled neurons were located within the PPN, DR, PAGvl, and CLI from bregma levels −7.90 to −6.65. a Within the PPN, most retrogradely labeled neurons (more ...)
Rostral to the midbrain CLI, CEAm- and/or BSTvl-projecting neurons were located within the ventral tegmental area (VTA; ) and the compact portion of the sub-stantia nigra (SNc; ). Retrograde labeling within the VTA was very sparse and was not quantified. The SNc contained a fair number of CEAm-projecting neurons (not quantified, ), but no BSTvl-projecting neurons.
Fig. 7 Distribution maps of retrogradely labeled neurons within the rostral midbrain/caudal forebrain. a–c Retrogradely labeled rostral midbrain neurons were located primarily within the SNc and VTA between bregma levels −5.65 and −5.00. (more ...)
Projections to the CEAm and BSTvl were found throughout the rostrocaudal extent of the thalamus (–) with large numbers of retrogradely labeled neurons within more caudally located thalamic nuclei (). In general, thalamic CEAm-projecting neurons were more prevalent than BSTvl-projecting neurons. At caudal levels, many CEAm-projecting and few BSTvl-projecting neurons were present within the auditory thalamus (Aud; ). In more rostral sections (), primarily CEA-projecting neurons were present within the medial parvicellular subparafascicular nucleus (SPFpm) and the parvicellular ventral posteromedial nucleus (VPMpc). Further rostrally, retrogradely labeled neurons extended more medially to join the midline thalamic nuclei group (MTN; ), including the central medial nucleus, intermediodorsal nucleus, and the PVT. CEAm- and BSTvl-projecting neurons were distributed throughout the rostrocaudal extent of the MTN (–), but were most numerous within the PVT. When the PVT was considered as a whole, there was no significant difference between the number of CEAm- versus BSTvl-projecting neurons (618 ± 117 vs. 646 ± 35 neurons; P = 0.83). Two-way ANOVA revealed a significant effect of PVT rostrocaudal level [F(1, 83) = 2.75, P < 0.01] and an interaction between tracer target site and PVT rostrocaudal level [F(13, 83) = 6.45, P < 0.001]. When the PVT was divided into three rostrocaudal segments, there was a clear difference in the distribution of CEAm- versus BSTvl-projecting neurons across the caudal, middle, and rostral thirds of the PVT (cPVT = caudal to bregma level −3.25; mPVT = bregma levels −2.85 through −2.00; rPVT = rostral to bregma level −2.00; ). Significantly larger numbers of BSTvl-projecting neurons were located within the rPVT compared to either the mPVT (310 ± 18 vs. 157 ± 7 neurons, P < 0.005) or the cPVT (310 ± 18 vs. 179 ± 12 neurons, P < 0.005). Conversely, the number of CEAm afferents within the cPVT was significantly greater than within the mPVT (320 ± 47 vs. 162 ±31 neurons, P < 0.05), but was not greater compared to the number of CEAm afferents in the rPVT (320 ± 47 vs. 196 ± 19 neurons, P = 0.72). Further analysis of retrograde labeling across the three rostrocaudal PVT levels revealed that the cPVT contained significantly greater numbers of CEAm afferents compared to BSTvl afferents (320 ± 47 vs. 179 ± 12 neurons, P < 0.05), while the rPVT contained a significantly greater number of BSTvl afferents compared to CEAm afferents (310 ± 18 vs. 135 ± 42 neurons, P = 0.02). The numbers of CEAm and BSTvl afferents within the mPVT were not significantly different (162 ± 31 vs. 157 ± 7 neurons, P = 0.87).
Fig. 9 Distribution maps of retrogradely labeled forebrain neurons at the level of the preoptic hypothalamus. Retrogradely labeled neurons were located within the SI, IPAC, BST, SFO, and LS of the basal forebrain; the MPO of the hypothalamus, and the DI/AI region (more ...)
Fig. 8 Distribution maps of retrogradely labeled forebrain neurons at the level of the tuberal hypothalamus. Retrogradely labeled neurons were located primarily within the CEA, BMA, and BLAp of the amygdala; within the LHA, VMH, and ARC of the hypothalamus; (more ...)
Hypothalamic regions generally contained many BSTvl-projecting neurons and smaller numbers of CEAm-projecting neurons. The hypothalamic distribution of these populations rarely overlapped, and therefore, were plotted (see , ) but not quantified. The density of BSTvl-projecting neurons in the hypothalamus appeared greatest within the lateral hypothalamic area (LHA; ) and the medial preoptic area (MPO; ). However, one exception to the largely separate hypothalamic distribution of BSTvl- and CEAm-projecting neurons was the parasubthalamic nucleus (PSTN; , ), which contained similar numbers of CEAm- and BSTvl-projecting neurons (133 ± 54 vs. 136 ± 11 neurons, P = 0.96). Interestingly, despite the dense overlapping distribution of CEAm- and BSTvl-projecting PSTN neurons, relatively few were double-labeled (10 ± 4 neurons, 4 ± 2% of total; ).
Within the amygdala, large numbers of CEAm- and BSTvl-projecting neurons were located within the posterior basolateral amygdala (BLAp; , , ) and basomedial amygdala (BMA; , ), while relatively fewer retrogradely labeled neurons were present within the lateral or medial amygdala (). There was a non-significant trend towards higher numbers of CEAm-versus BSTvl-projecting BLAp neurons (689 ± 155 vs. 388 ± 76 neurons; P = 0.16). Collateralized projections of BLAp neurons to both the CEAm and BSTvl were relatively common. Approximately one-third of BSTvl-projecting neurons and 20% of CEAm-projecting neurons within the BLAp were double labeled (13 ± 2% of total retrogradely labeled neurons, ). In contrast, despite similarly large numbers of CEAm- and BSTvl-projecting neurons within the BMA (877 ± 334 vs. 413 ± 151 neurons; P = 0.27; ), smaller proportions of these neurons were double-labeled as compared to the BLAp (6 ± 2% of total retrogradely labeled neurons, ).
As expected, iontophoresis of retrograde tracer into the CEAm produced abundant retrograde labeling within the BSTvl, and vice versa, as well as retrograde labeling within other regions of the extended amygdala (, , , ). CEAm-projecting neurons were located throughout the lateral BST, and BSTvl-projecting neurons were found throughout the CEAm and CEAl. Additional CEAm- and BSTvl-projecting neurons were scattered throughout the substantia innominata (SI; ), a region interposed rostrocaudally between the CEA and BST and described as part of the “central extended amygdala” (de Olmos and Heimer 1999
). In addition, a dense cluster of CEAm- and BSTvl-projecting neurons was located within a discrete subregion of the SI called the interstitial nucleus of the posterior limb of the anterior commissure (IPAC; , ) (Shammah-lagnado et al. 2001
). Quantification of retrogradely labeled IPAC neurons revealed no significant difference between the number of CEAm and BSTvl afferents (227 ± 73 vs. 262 ± 48 neurons; P = 0.71). A moderate proportion of IPAC neurons projected axons to both the CEAm and BSTvl (). The caudal part of the nucleus accumbens shell (ACBsh), particularly its dorsal medial tip, contained large numbers of BSTvl-projecting neurons but relatively few CEAm-projecting neurons ().
Fig. 11 Distribution maps of retrogradely labeled neurons within the rostral forebrain. Retrograde labeling within the basal forebrain was located primarily within the ACBsh and LS, and cortical labeling was located within DI/AI and Ila. The ACBsh and LS contained (more ...)
Three major regions of the cerebral cortex contained CEAm- and BSTvl-projecting neurons. The densest cortical input to the CEAm and BSTvl arose from a region situated near the rhinal sulcus, including the agranular and dysgranular insular cortex (AI and DI, respectively; , , ). Within the AI, significantly greater numbers of retrogradely labeled neurons projected to the CEAm versus the BSTvl (868 ± 208 vs. 102 ± 45 neurons; P = 0.02). More than 30% of BSTvl-projecting AI neurons also projected to the CEA (i.e., were double-labeled), whereas double-labeled neurons comprised only 3% of all AI input to the CEAm ().
A dense overlap of CEAm- and BSTvl-projecting neurons was observed within deep layers of the caudal infralimbic cortex (ILA; , ), with only a few scattered cells observed in the more dorsally situated prelimbic cortex (PL; , ). Quantification of ret-rogradely labeled neurons within the ILA revealed similar numbers of CEAm- and BSTvl-projecting neurons (96 ± 37 vs. 129 ± 43 neurons; P = 0.6). Despite their overlapping distributions, BSTvl- and CEAm-projecting neurons within the ILA rarely collateralized to both the CEAm and BSTvl (3 ± 2% of total retrogradely labeled neurons; ).
Within the ventral cerebral cortex, dense retrograde labeling was located within the postpiriform transition area (TR, sometimes referred to as the amygdala-piriform transition area; , ), with fewer retrogradely labeled neurons scattered throughout the more medially situated posterior amygdala (PA), and also within area CA1 of the ventral hippocampus (). Quantification of retrogradely labeled TR neurons revealed a significantly greater number of CEAm- versus BSTvl-projecting neurons (682 ± 94 vs. 192 ± 63 neurons; P = 0.01). Relatively few TR neurons collateralized to innervate both the CEAm and BSTvl (2 ± 1% of total retrogradely labeled neurons; ).
Retrograde labeling patterns after tracer iontophoresis into regions adjacent to CEAm and BSTvl
The goal of this study was to target specific subregions of the central extended amygdala, i.e., the BSTvl and the CEAm, for retrograde tracing of their afferent inputs. This clearly is a challenging task, given the relatively small size of each structure. Thus, we sought to determine whether tracer delivery sites that were viewed as “accurately centered” and relatively confined to the BSTvl or CEAm produced retrograde labeling that was distinct from labeling produced by tracer delivery centered in adjacent “incorrect” regions. “Incorrect” tracer delivery sites located adjacent to the BSTvl included the ventral pallidum (VP, lateral to the BST) and the posterior BST (pBST). Iontophoretic delivery of tracer into the VP produced abundant retrograde labeling in the medial subthalamic nucleus (STN), whereas accurate BSTvl iontophoretic delivery produced retrogradely labeled neurons in the adjacent PSTN with no labeling in the STN. Iontophoretic delivery of tracer into the pBST produced dense retrograde labeling within the MEA, whereas MEA labeling was much more sparse after accurate tracer placement in the BSTvl. Tracer delivery into the VP or pBST also produced little or no retrograde labeling within other brain regions that contained labeled neurons after accurate BSTvl tracer delivery, including the NTS, VLM, BLAp, TR, and PSTN.
“Incorrect” tracer delivery sites adjacent to the CEAm included the dorsally situated amygdala-striatal transition area (AStr), the CEAl, and/or the BLA. In contrast to labeling produced by accurate CEAm-targeted sites, iontophoretic delivery of tracer into the BLA produced substantially more retrograde labeling within the pontine locus coeruleus (Asan 1998), and bilateral retrograde labeling of neurons within layer 3 of the nucleus of the lateral olfactory tract, consistent with previous findings (Santiago and Shammah-Lagnado 2004
). Tracer delivery into the AStr retrogradely labeled neurons within secondary somatosensory cortex, which does not project to the CEA (Shammah-Lagnado et al. 1999
). This cortical region was not labeled in rats with accurate CEAm tracer delivery sites in the present study. Cases in which the center of the iontophoretic delivery site was located within the CEAl rather than the CEAm produced little or no retrograde labeling within the NTS or VLM, in contrast to results following CEAm-centered tracer delivery, and consistent with the preferential distribution of NA fibers within the CEAm as compared to the CEAl (shown in ). NA inputs to the CEA arise primarily from the caudal medulla, and the large majority of NTS and VLM neurons that project to the CEA are NA neurons (Myers and Rinaman 2002