Stefanie Roder

Collateral axonal projections to limbic structures from ventrolateral medullary A1 noradrenergic neurons.

Experiments were done to investigate whether catecholaminergic neurons within the ventrolateral medulla (VLM) send collateral axonal projections to the central nucleus of the amygdala (ACe) and the bed nucleus of the stria terminalis (BST). Unilateral microinjections of the fluorescent retrograde tracers fluorogold (FG) or rhodamine labelled latex micro-beads (Rd) were made into either ACe or BST in the rat. Brainstem sections were then processed immunohistochemically for the identification of cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase, dopamine beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT). Retrogradely labelled cell bodies projecting to either ACe or BST were found throughout the rostrocaudal extent of VLM, bilaterally. Approximately 44% of these retrogradely labelled neurons were found to contain both retrograde tracers. In addition, approximately 91% of the VLM neurons that send collateral axonal projections to ACe and BST were also immunoreactive to DBH. None were found to contain PNMT immunoreactivity. These results demonstrate that noradrenergic neurons of the A1 cell group in VLM innervate ACe and BST via collateral axonal projections and suggest that these VLM neurons may be directly involved in relaying cardiovascular afferent and/or visceral afferent information directly to these limbic structures.

Contribution of caudal ventrolateral medulla to the cardiovascular responses elicited by activation of bed nucleus of the stria terminalis

The contribution of caudal ventrolateral medulla (cVLM) to the mean arterial pressure (MAP) and heart rate (HR) responses elicited by microinjections of L-glutamate (GLU) into the cardiovascular responsive region of bed nucleus of the stria terminalis (BST) was investigated in the chloralose-anesthetized, paralysed and artificially ventilated rat. Unilateral injections of GLU into BST elicited decreases in MAP of -25 +/- 3 mmHg (n = 10) and HR of -13 +/- 3 bpm (n = 10). These cardiovascular responses were not altered after a 100 nl microinjection of 0.9% NaCl into cVLM. However, the magnitudes of the decreases in MAP and HR were attenuated (-11 +/- 3 mmHg and HR, -4 +/- 1 bpm, respectively) 5 min after a 100 nl microinjection of the reversible synaptic blocker cobalt chloride (CoCl2) into cVLM. Restimulation of BST 40 min after the 100 nl microinjection of CoCl2 in cVLM elicited cardiovascular responses that were not significantly different in magnitude from those evoked before the microinjection of CoCl2 (MAP, -23 +/- 4 mmHg; HR, -12 +/- 5 bpm). In an additional series of experiments (n = 3), restimulation of BST 1 h after an ipsilateral electrolytic lesion in cVLM elicited decreases in MAP (-11 +/- 2 mmHg) and HR (-8 +/- 4 bpm) that were significantly smaller than those elicited prior to cVLM lesion. These data suggest that a component of the pathways originating in BST involved in mediating depressor responses and cardiac slowing relays in cVLM.

Contribution of bed nucleus of the stria terminalis to the cardiovascular responses elicited by stimulation of the amygdala

Anatomical and physiological studies were done in the rat to investigate the possibility that the cardiovascular responses elicited by stimulation of central nucleus of the amygdala (ACe) were mediated via projections to bed nucleus of the stria terminalis (BST). In the first series, to determine the distribution of neurons in ACe that projected to the cardiovascular region of BST, the retrograde tracer Fluorogold (FG) or rhodamine latex micro-beads (Rd) were injected into BST. FG and Rd injections that overlapped the cardiovascular region of BST resulted in retrogradely labelled neurons throughout the amygdala. In ACe, retrogradely labelled neurons were observed primarily in the lateral subdivision of the rostral ACe compared to the caudal ACe. The medial subdivision of ACe was found to have very few retrogradely labelled neurons. In the second series, the effect of either blocking synaptic transmission in BST with CoCl2, chemical lesions of BST with ibotenic acid (IBO), or electrolytic lesions of BST on the depressor response elicited by either electrical or chemical stimulation of ACe was investigated in the chloralose-anesthetized, artificially ventilated and paralysed rat. Microinjections of CoCl2 into BST significantly attenuated the depressor responses to stimulation of the rostral components of the lateral subnucleus of ACe, but not those to stimulation of the caudal and medial components of ACe. Microinjections of IBO into BST or electrolytic lesions of BST resulted in similar effects on the depressor responses to ACe stimulation. Taken together, these data indicate that neurons within the rostral components of the lateral subnucleus of ACe project to the cardiovascular region of BST and mediate in part the depressor responses to stimulation of the rostral ACe. On the other hand, the depressor responses elicited from the caudal ACe are not mediated through BST. These results suggest that at least two independent pathways originate in the ACe that influence the circulation.

Collateral axonal projections from ventrolateral medullary non-catecholaminergic neurons to central nucleus of the amygdala

Retrograde tract-tracing techniques were used to investigate whether catecholaminergic neurons in the ventrolateral medulla (VLM) send collateral axonal projections to both central nuclei of the amygdala (ACe) in the rat. Rhodamine-labelled latex microspheres or fluorogold (2%) were microinjected into the region of either the right or left ACe. After a survival period of 10-12 days, the rats were sacrificed and transverse sections of the brainstem were processed immunohistochemically for the identification of cell bodies containing the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) or phenylethanolamine-N-methyltransferase (PNMT). Neuronal perikarya containing the retrogradely transported tracers were observed throughout the rostrocaudal extent of VLM, bilaterally. Approximately 10% of the retrogradely labelled neurons were observed to contain both retrograde tracers. The majority (79 +/- 6.8%) of these double labelled neurons were located within the caudal VLM and their number decreased rostrally. In addition, the proportion of double labelled neurons to single labelled neurons in VLM decreased rostrally; approximately 11% in the caudal VLM and 6% in the rostral VLM. Furthermore, approximately 21% of all VLM neurons that projected to ACe were found to be catecholaminergic: 75% of these were immunoreactive to TH and 25% to PNMT. However, no neurons were found in VLM that contained both retrograde tracers and immunoreactivity to TH or PNMT. These data demonstrate that axons originating from non-catecholaminergic neurons in VLM bifurcate to innervate ACe bilaterally. Although the function of these VLM neurons that project to both ACe is not known, they may be the anatomical substrate by which VLM neurons relay simultaneously autonomic and/or visceral sensory information to influence the activity of ACe.

GABAergic effects on the depressor responses elicited by stimulation of central nucleus of the amygdala

GABAergic inputs have been demonstrated in the central nucleus of the amygdala (ACe). However, the contribution of these inhibitory inputs to the cardiovascular responses elicited from the ACe is not known. Experiments were done in chloralose-anesthetized, paralyzed, and artificially ventilated male Wistar rats to investigate the effects of microinjections of GABA, the selective GABAA-receptor antagonist bicuculline, or the GABAB-receptor antagonist phaclofen, in the ACe on the mean arterial pressure (MAP) and heart rate (HR) responses elicited byl-glutamate (Glu) stimulation of the ACe. Microinjections of Glu in the ACe elicited decreases in MAP (-13.7 ± 1.6 mmHg) and HR (-5.3 ± 1.9 beats/min). The MAP and HR responses elicited by Glu stimulation of the ACe were significantly reduced (89%) by the prior microinjection of GABA in the same ACe site. In addition, at some sites in the ACe at which microinjection of Glu did not elicit depressor responses, Glu injections in the presence of phaclofen elicited decreases in MAP (-9.5 ± 1.0 mmHg) and variable changes in HR. On the other hand, the magnitude of the depressor responses elicited during stimulation of the ACe site in the presence of bicuculline was significantly attenuated (60%), whereas phaclofen had no effect on the magnitude of the depressor responses elicited by Glu stimulation of the ACe. These data suggest that GABAergic mechanisms in the ACe alter the excitability of ACe neurons involved in mediating changes in systemic arterial pressure and HR.

Caudal ventrolateral medullary projections to the nucleus of the solitary tract in the cat

The projections of neurons, in and around the A1 noradrenergic cell group of the caudal ventrolateral medulla (VLM), to nucleus of the solitary tract (NTS) were studied in the cat using the anterograde transport of Phaseolus vulgaris leucoagglutinin (PHA-L). PHA-L was micro-iontophoresed into the region of the A1 noradrenergic cell group and after a 7-17 day survival period animals were sacrificed and brainstem sections were processed for PHA-L or tyrosine hydroxylase (TH) immunoreactivity. PHA-L injections within the region of the A1 cell group resulted in labelled fibers with their presumptive terminal boutons primarily in the ipsilateral commissural and medial subnuclei of NTS. A light projection to the ipsilateral parvocellular lateral and ventrolateral subnuclei of the NTS complex was also observed. These data demonstrate that neurons in the region of the A1 noradrenergic cell group project to regions of NTS that receive cardiovascular afferent inputs and suggest that VLM may influence the activity of neurons in NTS involved in the reflex regulation of the circulation.

Effect of noradrenergic inputs on the cardiovascular depressor responses to stimulation of central nucleus of the amygdala

Experiments were done in chloralose anesthetized, paralyzed and artificially ventilated male Wistar rats to investigate the effects of microinjections of either norepinephrine (NE) or tyramine into the central nucleus of the amygdala (ACe) on the arterial pressure (AP) and heart rate (HR) responses elicited by glutamate (Glu) stimulation of the ACe. Microinjections of Glu into the ACe elicited decreases in mean AP (-23+/-3 mmHg) and HR (-11+/-3 bpm). Microinjections of NE or tyramine into these sites did not elicit cardiovascular responses. However, Glu into the ACe in the presence of NE or tyramine elicited depressor or bradycardic response that were significantly smaller (70-100%) in magnitude than to Glu alone. These data suggest that noradrenergic mechanisms in the ACe alter the excitability of ACe neurons involved in mediating changes in systemic AP and HR.

Direct projections from caudal ventrolateral medullary depressor sites to the subfornical organ

Experiments were performed in the male Wistar rat to investigate the projections from cardiovascular responsive sites in the ventrolateral medulla (VLM) to the subfornical organ (SFO). Unilateral iontophoretic injections of Phaseolus vulgaris leucoagglutinin (PHA-L) were made into either caudal VLM (CVLM) sites at which microinjection of l-glutamate (10 nl; 0.25 M) elicited decreases in mean arterial pressure or into rostral VLM (RVLM) sites at which l-glutamate microinjection elicited increases in arterial pressure. After a survival period of 7-10 days, transverse sections of the forebrain and brainstem were processed for PHA-L immunoreactivity. After injections of PHA-L into the CVLM, axonal and presumptive terminal labeling was found bilaterally throughout the rostrocaudal extent of the SFO, although most of the projections were observed within the rostral half of the nucleus. Within the SFO, labeling was found primarily in the lateral aspects of the nucleus, often in close proximity to blood vessels. In addition, CVLM injections resulted in labeling within the organum vasculosum of the laminae terminalis (OVLT) and within the ventral and dorsal components of the median preoptic nucleus (MnPO) bilaterally, but with an ipsilateral predominance. In contrast, PHA-L injections into the RVLM did not result in axonal labeling in the SFO or OVLT, although a few labeled axons were found to course through the region of the ventral component of MnPO. These data have demonstrated that neurons within the cardiovascular responsive region of the CVLM send direct axonal projections to the SFO and other structures of the laminae terminalis, and suggest that the CVLM may function in the modulation of the activity of neurons of circumventricular organs to intra- and extracellular signals of body fluid balance.

17β-Estradiol alters the response of subfornical organ neurons that project to supraoptic nucleus to plasma angiotensin II and hypernatremia

This study was done in urethane anesthetized, ovariectomized (OVX) female rats that were either implanted or not implanted with silastic capsules containing17β-estradiol (E2) to investigate the effect of systemic changes in E2 on the discharge rate of subfornical organ (SFO) neurons that projected to supraoptic nucleus (SON) and responded to changes in plasma levels of angiotensin II (ANG II) or hypernatremia. Extracellular single unit recordings were made from 146 histologically verified single units in SFO. Intra-carotid infusions of ANG II excited ~57% of these neurons, whereas ~23% were excited by hypertonic NaCl. Basal discharge rate of neurons excited by ANG II or hypertonic NaCl was significantly lower in OVX+E2 rats compared to OVX only animals. The response of SFO neurons antidromically activated by SON stimulation to intra-carotid injections of ANG II or hypertonic NaCl was greater in the OVX only compared to the OVX+E2 rats. Intra-carotid injections of E2 in either group attenuated not only the basal discharge of these neurons, but also their response to ANG II or hypertonic NaCl. In all cases this inhibitory effect of E2 was blocked by an intra-carotid injection of the E2 receptor antagonist ICI-182780, although ICI-182780 did not alter the neurons response to ANG II or hypertonic NaCl. Additionally, ICI-182780 in the OVX+E2 animals significantly raised the basal discharge of SFO neurons and their response to ANG II or hypertonic NaCl. These data indicate that E2 alters the response of SFO neurons to ANG II or NaCl that project to SON, and suggest that E2 functions in the female to regulate neurohypophyseal function in response to circulating ANG II and plasma hypernatremia.

Convergence of ventrolateral medulla and aortic baroreceptor inputs onto amygdala neurons

Experiments were done to investigate the effect of stimulation of neurons in ventrolateral medulla (VLM) and aortic baroreceptors on the discharge rate of amygdala neurons. The region of central nucleus of the amygdala (ACe) was explored for spontaneously active single units that altered their discharge rate to electrical stimulation of VLM in the alpha-chloralose anesthetized rat. Responsive units were also assessed for their response to electrical stimulation of the aortic depressor nerve (ADN). Stimulation of VLM altered the discharge rate of 47% (43/92) of the units tested in and around the region of ACe. Of these units, 60% (26/43) were excited (mean latency, 13.6 +/- 3.6 ms) and 40% (17/43) were inhibited (mean latency, 23.1 +/- 4.3 ms) by VLM stimulation. Of the 43 units that responded to stimulation of VLM, 19 (44%) also responded to ADN stimulation with a mean latency of 32.5 +/- 7.6 ms. These data demonstrate that inputs from VLM and ADN converge onto ACe neurons and suggest that VLM may function as a relay for cardiovascular afferent information to the amygdala.