Electrophysiology of the Circumventricular Organs

FRONTIERS IN NEUROENDOCRINOLOGY ARTICLE NO.

17, 440–475 (1996)

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Electrophysiology of the Circumventricular Organs ALASTAIR V. FERGUSON

AND

JAIDEEP S. BAINS

Department of Physiology, Queen’s University, Kingston, Ontario, Canada K7L 3N6

Since the first anatomical description of the circumventricular organs (CVOs) as a structurally distinct group of regions in the central nervous system (CNS), considerable information has implicated these structures as physiologically significant autonomic control centers located at the blood–brain interface. Specialized features of these structures, such as their extensive vasculature, lack of the normal blood–brain barrier (BBB) (i.e., capillaries have a fenestrated endothelium), and dense aggregations of a variety of peptidergic receptors, support an involvement of the CVOs in communication between the circulation and the CNS. The two best understood examples of CVOs with the ability to sense circulating substances impermeable to the BBB are the subfornical organ (SFO) and the area postrema (AP). Specifically, the ability of numerous peptides to influence CNS function, as the result of actions on the neural substrate of these structures has been especially well documented. Considerable anatomical, biochemical, pharmacological, and physiological evidence has implicated these structures as CNS sites at which angiotensin (ANG), atrial natriuretic peptide (ANP), vasopressin (VP), and endothelin (ET) act to influence neuroendocrine and other more classical autonomic functions. In the following sections, we review neurophysiological studies which have provided new and exciting insights regarding the specific neural pathways and cellular mechanisms through which CVO neurons are able to exert their profound influences over central autonomic control. r 1996 Academic Press, Inc.

INTRODUCTION

The dominant role of the brain in the hierarchical control of the autonomic nervous system demands that it receives extensive afferent information regarding the milieu interieur. This information is derived from two primary sources: (1) peripheral and visceral sensory systems which transmit information through classical sensory pathways into the central nervous system (CNS)1 and (2)

Address reprint requests to Alastair Ferguson. Fax: (613) 545-6880. 1 Abbreviations used: 4-AP, 4-aminopyridine; A1-NA, A1 area of the nucleus ambiguus; aCSF, artificial cerebrospinal fluid; ANG, angiotensin; ANP, atrial natriuretic peptide; AP, area postrema; AV3V, anteroventral third ventricle; BBB, blood–brain barrier; CCK, cholecystokinin; CGRP, calcitonin gene-related peptide; CNS, central nervous system; CVO, circumventricular organ; DMV, dorsal motor nucleus of the vagus; ET, endothelin; GnRH, gonadotropin-releasing hormone; HRP, horseradish peroxidase; IA, A-current; IK, delayed rectifier; IML, intermediolateral cell column; LH, luteinizing hormone; LTS, low threshold spikes; MnPO, median preoptic nucleus; MS-DBB, medial septum-diagonal band of broca; NTS, nucleus tractus solitarius; OVLT, organum vasculosum of the lamina terminalis; OXY, oxytocin; PBN, parabrachial nucleus; PVN, paraventricu0091-3022/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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sensory systems in the brain which monitor the constituents of the circulation to assess the physiological status of the individual. However, in view of its protected position behind the blood–brain barrier (BBB), the CNS is theoretically unable to monitor many of the most significant controlled variables which constitute this internal environment (osmolarity, hydrophilic amino acid concentrations, and peptide concentrations). In effect, the BBB which acts to protect the brain from large shifts in these variables (their fine control is a prerequisite for normal CNS function) theoretically also precludes CNS monitoring of such essential information regarding the physiological status of the internal environment. The logic of such a system is clear, provided the brain can gain access to essential sensory information through alternative mechanisms. The circumventricular organs (CVOs), comprising the subfornical organ (SFO), area postrema (AP), organum vasculosum of the lamina terminalis (OVLT), median eminence, and neurohypophysis, are such a specialized group of CNS structures, which lack the normal BBB, and thus provide such an alternative. Within these structures, circulating substances can directly access CNS tissue, and they thus play a pivotal role in blood–brain communication. Since the first anatomical description of the CVOs as a structurally distinct group of regions in the CNS, considerable information has implicated these structures as physiologically significant autonomic control centers located at the blood–brain interface. The majority of this work, and thus the primary focus of this review, has been directed toward the SFO, AP, and OVLT, although the median eminence and neurohypophysis are also mentioned briefly. Specialized features of these structures, such as their extensive vasculature, lack of the normal BBB (i.e., capillaries have a fenestrated endothelium), and dense aggregations of a variety of peptidergic receptors, support an involvement of the CVOs in communication between the circulation and the CNS (30, 39, 59, 60, 75). Such information transfer could be from blood to neuron, from neuron to blood, or conceivably between cerebrospinal fluid and either the circulation or neurons. The median eminence and neurohypophysis provide persuasive examples of CVOs in which the primary direction of communication is from neural tissue (hypothalamic neurosecretory neurons) to the circulation. Within such a framework, the lack of normal BBB presumably facilitates diffusion of released hypothalamic peptides from axonal terminals into the blood stream following release. In contrast, perhaps the most significant functional consequence of this lack of a normal BBB in other CVOs is that it permits circulating substances which would not normally cross the barrier to directly access neural tissue within these structures. The two best understood examples of CVOs with the ability to sense circulating substances impermeable to the BBB are the SFO, located in the forebrain, and the medullary AP. Specifically, the ability of numerous peptides to influence lar nucleus; RVLM, rostral ventrolateral medulla; SFO, subfornical organ; SHR, spontaneously hypertensive; SON, supraoptic nucleus; TEA, tetraethylammonium; TTX, tetrodotoxin; VP, vasopressin; WKY, wistar kyoto.

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CNS function, as the result of actions on the neural substrate of these structures, has been especially well documented. Considerable anatomical, biochemical, pharmacological, and physiological evidence has implicated these structures as CNS sites at which angiotensin (ANG), atrial natriuretic peptide (ANP), vasopressin (VP), and endothelin (ET) act to influence neuroendocrine and other more classical autonomic functions. Much of this evidence has been elegantly described in a number of review articles (4, 8, 9, 60, 75) and therefore does not represent the major focus of this review. Rather, in the following sections, we will concentrate on neurophysiological studies directed toward understanding: x the intrinsic properties of CVO neurons and nerve terminals, x the integrated role of neural and humoral input in controlling the activity of these neurons, x the identified efferent projections of CVO neurons which endow on these structures their ability to control autonomic output and neurosecretion.

CVOS OF THE LAMINA TERMINALIS

The forebrain CVOs have been viewed historically as structures involved primarily in the central regulation of body fluid balance (75, 139, 156, 157). Thus the majority of work seeking to understand the role of these unique structures has focused on their ability to sense changes in the chemical constituents of the systemic circulation (e.g., concentration of ANG or osmolarity), which are important mediators of a systemic response to shifts in fluid volume (13, 50, 90). The primary and secondary efferent connections of these structures with hypothalamic neurosecretory nuclei, and autonomic nuclei in the brain stem and spinal cord, respectively, serve to highlight their ability to translate an incoming hormonal message into neural signals which control the output of autonomic control centers within the CNS. Although the role of these CVOs in the regulation of fluid balance has received the lion’s share of recent experimental attention, an increasing body of evidence now suggests these structures may make important contributions in a number of other integrated CNS responses.

SUBFORNICAL ORGAN

The SFO, which protrudes into the third ventricle, is much like the other CVOs in that it is highly vascularized (30, 169). Morphologically, it can be divided into three distinct zones, with the largest or central region consisting exclusively of neuronal cell bodies and glial cells, whereas the rostral and caudal areas contain primarily nerve fibers with very few neurons and glial

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cells (30). Anatomical studies, using tritiated amino acids (104) as well as both anterograde and retrograde tracing methods (94, 170), have described prominent SFO efferent projections to the anteroventral third ventricle (AV3V) and the hypothalamus. Within the AV3V region, these fibers terminate in the median preoptic area, the OVLT, and the adjacent periventricular nucleus. The hypothalamic projections terminate in the supraoptic (SON) and paraventricular nuclei (PVN), suggesting their involvement in the regulation of oxytocin (OXY) and VP secretion, as well as their potential to modify autonomic output through descending projections from PVN to the medulla and spinal cord. Some neurons in SFO provide collateral input to both SON and PVN (170), raising the possibility that they may be capable of eliciting integrated autonomic and neuroendocrine responses from these structures. Often overlooked in the haste to assign a role for SFO exclusively in body fluid homeostasis are the efferent projections of this structure to the infralimbic cortex, the rostral and ventral parts of the bed nucleus of the stria terminalis, the zona incerta, and the dorsal and median raphe nuclei (89, 148). However, the functional significance of these projections is not yet understood. The afferent projections to SFO, although neither as prominent nor as well studied as its efferent fibers, still provide input from a number of CNS structures, including the median preoptic nucleus (MnPO), nucleus reuniens of the thalamus (93), nucleus tractus solitarius (NTS) (177), lateral hypothalamus (93), and midbrain raphe (89).

Intrinsic Properties of SFO Neurons

The intrinsic properties of SFO neurons have been explored in both CNS slice preparations using extracellular and whole cell recording techniques and in dissociated cell preparations using whole cell recordings. Extracellular recordings have demonstrated that the majority of SFO neurons recorded in vitro display a continuous or irregular pattern of spontaneous action potentials, although a small percentage of cells do show short bursts in activity lasting less than 2 s (110). In vivo extracellular recordings have also shown minimal (,1.0 Hz) spontaneous activity in the majority of SFO neurons (164, 173). Some SFO neurons also display a ‘‘notch’’ on the falling phase of the action potential (110), a phenomenon observed with some frequency in the magnocellular neurons of the SON. Current clamp observations from both coronal slices (A. V. Ferguson and Z. Li, submitted for publication), and dissociated cells (A. V. Ferguson et al., submitted for publication), indicate that SFO neurons have resting membrane potentials in the 257- to 265-mV range and exhibit an irregular firing pattern with none of the bursting activity which is prevalent among neurons from other hypothalamic regions. Many SFO neurons recorded from our slice preparations exhibit marked synaptic activity (Fig. 1), with both excitatory and inhibitory postsynaptic potentials often exceeding 5 mV. Neither the chemical nature nor the anatomical origin of these inputs has been characterized.

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The input resistance of SFO neurons is apparently very high, often exceeding 1 gigaohm (A.V. Ferguson and Z. Li, in press), suggesting that the dendritic tree is likely very compact. This finding has been confirmed by our lucifer yellow fills of these cells which show an ovoid cell body of 10–15 µm in diameter and one perhaps two axons, but very few dendritic arborizations (Fig. 1). When hyperpolarizing current pulses are injected into cells at resting membrane potential, a delayed return to baseline is often observed, indicating the presence of a rapidly activating and inactivating potassium current, a finding which has been confirmed by our voltage clamp recordings (A.V. Ferguson and Z. Li, in press; A.V. Ferguson et al., submitted for publication). In response to depolarizing pulses, SFO neurons can either fire a single spike or respond with a burst of action potentials at frequencies greater than 30 Hz. Although not always obvious, some degree of spike-frequency adaptation is also observed during the burst sequences (Fig. 1). Not surprisingly, SFO neurons which display prominent action potentials also possess large, rapidly activating, voltage-dependent sodium currents (A.V. Ferguson et al., submitted for publication). The complete abolition by tetrodotoxin (TTX) of both this current and action potentials in current clamp mode suggests that this sodium current is responsible for the observed action potential. In the presence of TTX, depolarizing pulses reveal both a rapidly activating/inactivating current and a sustained outward current. Both currents are abolished when internal K1 is replaced by Cs1. In SFO slices, these currents demonstrated pharmacological similarity to the A current (reduced by 4-aminopyridine (4-AP)) and the delayed rectifier (blocked by tetraethylammonium (TEA)) (A.V. Ferguson and Z. Li, in press) responsible for the termination of the action potential and the subsequent after-hyperpolarization in other CNS tissue. Blockade of sodium channels by TTX and replacement of K1 with Cs1 also reveals the presence of a small, slowly activating, inward current in response to a depolarizing current step (A.V. Ferguson et al., submitted for publication). This current has qualities similar to other neuronal calcium currents, although both the definitive kinetic and the ionic characterization of this current await further experimentation. The congruence of observations obtained from recordings made in slice and dissociated cell preparations suggests that these dissociation techniques do not in themselves significantly alter the basic electrophysiological properties of these cells. In fact, following dissociation, adult SFO neurons maintained

FIG. 1. The intrinsic membrane properties of two different SFO neurons recorded in whole cell patch mode from a coronal slice. (a) Spontaneous action potentials and inhibitory postsynaptic potentials .5 mV are evident in the upper recording in which the cell is maintained at resting membrane potential. Hyperpolarizing the cell to 276 mV results in abolition of spontaneous action potentials and spontaneous IPSPs, suggesting these events were likely due to a GABA mediated Cl2 conductance. (b) This neuron demonstrates robust firing in response to depolarizing pulses of 120 and 130 pA. Notice the slowing of the discharge during the depolarizing pulses (spike frequency adaptation) and the after-hyperpolarization (arrow) which follows the burst. (c) Lucifer yellow fill shows a typical SFO neuron with an ovoid cell body (10–15 µm) with very few dendritic processes and two diverging axons (scale bar, 15 µm).

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under tissue culture conditions have been shown to maintain a consistent resting membrane potential and input resistance for up to 5 days (A.V. Ferguson et al., submitted for publication), suggesting that these cells have specialized features, which aid in their survival under these conditions. Such an observation is perhaps not surprising when one considers that under normal conditions SFO neurons must survive without the protective umbrella offered to all other CNS neurons by the BBB.

Circulating Factors and the Activity of SFO Neurons

Angiotensin A central site of ANG actions, through which this peptide acts to increase blood pressure, was first demonstrated by Bickerton and Buckley (30). A decade later, Felix and Akert (35) demonstrated that SFO neurons in the cat were responsive to ANG, a finding which was made more physiologically relevant by the demonstration that SFO was the CNS site through which ANG exerted its centrally induced pressor (97) and drinking responses (139). Since then, autoradiographic techniques have identified ANG receptors within SFO (102, 129), while the development of nonpeptidergic antagonists for these receptors has revealed that they are primarily of the AT1 subtype (56). These receptors are also apparently downregulated by marked sodium depletion (124), while stress can increase the density of ANG receptors in SFO (21). Clearly the presence of receptors suggests a physiological role for the agonist, and indeed circulating ANG has been shown to influence the activity of SFO neurons both in vivo (62, 153) and in vitro (87, 110, 134). The studies have clearly demonstrated that regardless of species (rat, cat, duck), the predominant effect of ANG on SFO neurons is to increase their activity. Approximately 70% of cells tested in SFO show an excitatory response to this peptide, a finding which is consistent from one study to the next. This response is unaffected by synaptic blockade, suggesting a direct action of ANG on the SFO neuron. This effect has now been shown to be the result of specific ANG actions at the AT1 receptor, since the excitation induced by ANG can be blocked in the presence of losartan, a specific AT1 receptor antagonist (87). Whole cell patch clamp recordings have provided us with more definitive information regarding the underlying mechanisms through which ANG exerts its primary excitatory effect on SFO neurons. In coronal SFO slices, ANG inhibits peak IA (A current), but has no effect on the sustained IK (delayed rectifier) (A.V. Ferguson and Z. Li, in press). This suggests that ANG may increase the firing of a cell which already shows spontaneous activity by minimizing the after-hyperpolarization, thereby maintaining the membrane potential closer to threshold. Current clamp recordings from dissociated cells, however, show that ANG depolarizes cells with a resulting decrease in input resistance (Ferguson et al., submitted for publication), as illustrated in Fig. 2. This suggests an increase in cationic influx, a finding which is at present not

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FIG. 2. The voltage responses of an SFO neuron to current pulses before and during administration of ANG are displayed on the left. The decrease in magnitude of the voltage traces following ANG indicates a decrease in input resistance. The depolarizing pulses after ANG are not of a sufficient magnitude for the cell to reach threshold, even though the cell is depolarized by 4 mV. The summary I-V plot of this cell, which demonstrates a dramatic increase in conductance, is on the right.

immediately reconcilable with the voltage clamp data from slices. It is conceivable that as the decrease in IA is small, its effects on input resistance may be masked in current clamp mode by a much larger influx of a cation such as calcium. The dissociated cells may in fact provide the method of choice for the study of SFO and other CVO neurons, since a majority of the input to these cells is from the circulation and not of a synaptic nature. Thus the dissociation of these neurons does not necessarily remove all of their normal afferent input. Atrial Natriuretic Peptides There is also an accumulating body of evidence that ANG effects on SFO neurons may be modulated by a second circulating peptide, namely atrial natriuretic peptide (ANP). Although there are an abundance of ANP receptors within SFO (122, 129), the peptide by itself was initially found to do little to the activity of SFO neurons (,20% affected). Observed effects were predominantly inhibitory, and since these effects were opposed to the actions of ANG, it was hypothesized that ANP may antagonize the excitatory effects of ANG on SFO neurons. Indeed this was found to be the case, as ANP application depressed ANG induced excitation by more than 40% in over 80% of the cells tested (65, 173). Physiological relevance for these findings was provided by the demonstration that ANP injected into SFO reduced drinking induced by ANG administra-

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tion into SFO (34). Recent evidence, however, suggests ANP does not antagonize the pressor response to ANG microinjection into SFO (72) and that ANP may independently decrease blood pressure. An opposing view of ANP actions on SFO neurons is offered by the work of Hubbard and colleagues with their demonstration that ANP excites up to 45% of SFO neurons in their slice preparation (15). This brief response is unaffected by synaptic blockade, suggesting that it is the result of direct ANP actions on the SFO neurons under investigation. Clearly, understanding these complex interactions between ANG and ANP occurring in single SFO neurons is an area of particular interest. The ability to identify the mechanisms through which known ligands influence specific ion channels and intracellular transduction mechanisms in SFO neurons represents a significant step toward an overall understanding of the integrative functions of this structure.

Other Peptides Although ANG has been the most thoroughly investigated peptide with regard to actions on SFO neurons, a number of other peptides can also influence the activity of these cells. Recent work suggests that the heptapeptide angiotensin III (ANG III) may also have excitatory effects on SFO neurons. This effect on SFO neurons is observed in both spontaneously hypertensive (SHR) and Wistar/ Kyoto (WKY) rats and can be blocked by an ANG III receptor antagonist (174). Binding studies also provide evidence regarding the presence of receptors for another potent circulating vasoconstrictor, endothelin (ET) (82, 109), within the SFO. ET binds to its receptors in SFO to increase circulating VP concentration and blood pressure (165), and following intravenous administration has also been shown to increase the firing frequency of the majority of SFO neurons antidromically identified as projecting to PVN (164). Some evidence also indicates that prostaglandins, specifically PGF2a , can also increase the activity of a small proportion of cells within SFO (72). This expanding field of work, based on the premise that circulating pyrogens affect the CNS by way of the CVOs, is discussed further in the following section on the OVLT. Recent work has also suggested that SFO may be an important site for the CNS actions of relaxin, an ovarian hormone present in large amounts in the circulation during the second half of gestation. Relaxin binding sites have been shown in SFO and also to a lesser degree in SON and PVN (115). Relaxin inhibits milk ejection in lactating rats by acting on SFO neurons (143), which in turn likely influence the activity of OXY neurons in SON and PVN (143). Relaxin also increases circulating concentrations of both VP and OXY and increases the firing rate of both VP and OXY neurons in lactating rats (168). Finally, relaxin, when injected into the cerebral ventricles, increases blood pressure, a response which is abolished by lesioning SFO (106). These early findings point to an important role for relaxin in the regulation of fluid levels and milk ejection through its actions at SFO. There is, however, a great deal of

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dissent with regard to the short-term versus long-term actions of this peptide. Clearly, this is a potentially exciting area which needs further exploration. Osmotic Changes The role of CNS structures as potential osmosensors has been debated for quite some time. The majority of evidence now supports the view that within the CNS there exist specific populations of neurons displaying nonspecific cationic conductances which are modulated by osmolarity. This intrinsic osmosensitivity which has been demonstrated to be a property of neurons in the SON, and more recently the OVLT and SFO, was the subject of a recent review (13). Consequently, the present section provides only a brief overview of the evidence implicating the SFO as a potential osmosensor. Attention has focused on the CVOs located in the lamina terminalis, since destruction of either of these regions results in a blunted response to systemic hypertonicity (98). The other obvious reason is due to the abundance of connections between both SFO and OVLT and the hypothalamic neurosecretory nuclei (27, 90, 104, 113, 121), suggesting these structures are capable of regulating the release of VP. The potential importance of SFO in any CNS response to osmotic challenge has been recently highlighted in studies utilizing proto-oncogenes such as c-fos and its protein product (FOS) as a marker for neuronal activation. Studies have demonstrated that an injection of hypertonic saline results in a marked increase in c-fos immunoreactive nuclei in SFO within 30 min of the osmotic challenge (58, 112). Interestingly, electrolytic lesions of SFO do not prevent osmotic activation of cells in PVN or SON, suggesting that SFO and the magnocellular neurosecretory cells are simultaneously but separately activated (58). In vivo single unit recording studies have also shown that SFO (62) and vasopressinergic magnocellular neurons in PVN (155) are activated by peripheral hypertonic saline, the latter effects being reversibly reduced (but not abolished) by microinjection of lidocaine into SFO (155), observations which imply that SFO efferents do in fact contribute a component of the osmotic response of PVN neurons. The role of SFO as an osmosensor has been confirmed by in vitro work demonstrating that SFO neurons show an increase or decrease in activity in response to hyper- or hypotonic stimuli, respectively (138). Interestingly, while the majority of cells show opposing responses to these two stimuli, some respond only to osmotic shifts in one direction or the other. This study also suggested that SFO neurons are intrinsically osmosensitive, since the majority of cells responded to osmotic challenge even in synaptic isolation. Preliminary observations on acutely dissociated SFO neurons confirm this hypothesis (13). Hypertonic stimulation of SFO neurons results in a reversible increase in membrane conductance and the appearance of an inward current reversing near 245 mV. These findings are similar to those observed in SON neurons, in which the response to hypertonicity is thought to be mediated by a nonspecific cationic conductance (114).

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FERGUSON AND BAINS Neural Inputs Controlling SFO Cell Activity

Although the multiple afferent inputs to SFO neurons derived from the systemic circulation have been a topic of considerable attention, the exploration of the roles of specific neural inputs in controlling the activity of SFO neurons has been sadly lacking. In vivo electrophysiology has demonstrated that SFO neurons receive primarily excitatory input from the lateral hypothalamic area (152), but this is one of the few examples of functional information regarding neural afferent input to SFO neurons. The paucity of identified anatomical sites which send significant neural input to the SFO represents the primary explanation for the absence of further data. Perhaps on this occasion, ‘‘absence of evidence should for the most part be interpreted as evidence of absence.’’

SFO Efferent Projections Involved in Neurosecretory and Autonomic Control

The actions of ANG in SFO to increase blood pressure can be eliminated by cutting the ventral stalk of SFO (91). In vivo, extracellular studies have demonstrated that ANG excites the majority of SFO neurons projecting to MnPO (153), supporting the hypothesis that drinking responses to increases in circulating ANG, although mediated via MnPO, are likely initiated through ANG actions at SFO. Neurons projecting from SFO to cell groups in PVN and SON are also influenced by ANG. Intracarotid ANG infusion excites 40% of the SFO neurons antidromically identified as projecting to PVN and 24% of those with identified projections to SON (62). The discrepancy between the two groups serves to highlight the differences between the two hypothalamic nuclei. Although both nuclei contain cells which synthesize and secrete OXY and VP, PVN contains additional cell groups which are responsible for the regulation of ACTH (149, 150) as a result of their connections with the median eminence, as well as sympathetic outflow through connections with cells located in autonomic centers in the brain stem and spinal cord (131, 132, 147). It is conceivable, then, that separate populations of cells within SFO, both activated by ANG, are responsible for the disparity in the observed responses. Electrical stimulation of SFO has been shown to increase the firing rate of putative vasopressinergic and oxytocinergic neurons in both SON (136) and PVN (43, 151). These data correlate well with studies demonstrating that such stimulation also results in increases in circulating concentrations of these peptides (44, 45). The magnocellular neurons have also been shown to be activated by circulating ANG, an effect which is abolished by SFO lesions (47), supporting the conclusion that the SFO represents the primary site of action for such effects of circulating ANG. However, perhaps the most fascinating feature of this particular projection of SFO neurons (known to be activated by circulating ANG) rests with the anatomical (92) and electrophysiological (74, 86) data in support of the conclusion that it also utilizes ANG as a neurotransmitter released from its nerve terminals in both SON and PVN.

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PVN neurons projecting to the dorsal medulla and the intermediolateral cell column (IML) in the spinal cord have been implicated in mediating at least the rapid component of the pressor response to SFO stimulation (61). Cells in PVN projecting to both of these structures are excited by electrical stimulation of SFO (2, 42), but only a small percentage of the cells within the medulla show a response to systemic ANG (37). The explanation for this lack of sensitivity of these caudally projecting PVN neurons is not immediately apparent, although it should be emphasized that there is no direct evidence demonstrating that pressor actions of ANG in SFO are dependent upon sympathetic activation. Stimulation studies similar to those described above have also been used to demonstrate that SFO provides indirect input to the hypothalamus via a projection to the medial septum–diagonal band of Broca (MS-DBB). Cells in MS-DBB antidromically identified as projecting SON or PVN receive predominantly excitatory input from SFO (31, 41). The physiological relevance of this projection is unclear, but it has been suggested to play a role in mediating the above-mentioned inhibitory input from SFO to SON, since administration of local anaesthetics directly into MS-DBB blocks the inhibitory effects of SFO stimulation on putative vasopressinergic cells (154). A bisynaptic pathway from SFO to PVN, with a relay in MnPO, has also been confirmed. Electrical stimulation of SFO or, alternatively, ANG actions at SFO excite approximately half of the MnPO neurons projecting to PVN (153). Along with its input to hypothalamic neurons projecting to the posterior pituitary, SFO also provides input to PVN neurons projecting to the median eminence. Although not as pronounced as the input to magnocellular cells in PVN, SFO still excites 30% of PVN cells antidromically identified as projecting to the median eminence, while inhibiting a smaller percentage (43). The cells excited by electrical stimulation of SFO are also excited by systemic ANG infusions (38), suggesting ANG may act through SFO to control secretion of anterior pituitary hormones. Additional evidence implicating the SFO in the control of these hormones can be derived from studies examining SFO input to MS-DBB neurons projecting to the median eminence. A high percentage of these putative gonadotropin-releasing hormone (GnRH) cells (85%) are excited by SFO stimulation (32), while electrical stimulation in SFO of conscious animals has been shown to increase circulating luteinizing hormone (LH) concentrations (33). Collectively these observations provide support for the hypothesis that SFO may also play some, as yet unidentified, role in the central control of reproductive function. The information described above presents a clear picture of the ability of the constituents of the general circulation to exert considerable control over the activity of SFO neurons, and thus to influence primary projection sites of these neurons as summarized in Fig. 3. However, the integrative functions of SFO, through which it potentially combines information regarding multiple aspects of the internal environment and initiates complex physiological responses, presents a significant challenge to future research.

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FIG. 3. The effects of different peptides on SFO neurons. The subsequent effects of activation (1 excitatory, 2 inhibitory) of these cells on the activity of both identified hypothalamic neurons are also highlighted.

ORGANUM VASCULOSUM OF THE LAMINA TERMINALIS

The OVLT is a midline structure located immediately dorsal to the optic chiasm. It is very similar to SFO in that its nonciliated ependymal surface protrudes into the third ventricle. Tracing studies using the retrograde tracer horseradish peroxidase (HRP) have demonstrated that OVLT receives input from SFO, the MnPO region, and the brainstem (121). Additional afferent input is provided by a number of structures located in the hypothalamus, including the ventromedial nucleus, the arcuate nucleus, the anterior and posterior hypothalamus, as well as cell groups located dorsolateral to SON (121). The use of tritiated amino acids has revealed that OVLT sends dense efferent projections to SON (90, 121). Additional fiber tracts terminate in the stria medullaris and the basal ganglia (121).

Intrinsic Properties of OVLT Neurons

A limited amount of work has been done to define the membrane properties of OVLT neurons. The difficulty associated with accurately defining the borders of

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this structure both in vivo and in vitro represents one of the reasons for this lack of information. Although in vitro extracellular recordings from AV3V neurons demonstrating that these cells show either continuous or irregular patterns of spontaneous activity (110, 173) probably include some recordings from OVLT neurons, these two structures clearly are not one and the same thing. Thus this information should not be over interpreted. Sharp electrode recordings have been obtained specifically from OVLT neurons in brain slices (108) and have demonstrated that these neurons have resting membrane potentials in the range 250 to 267 mV (mean: 260.9 6 1.0 mV). Their input resistances range from 65 to 360 MV (mean: 172 6 18.6 MV), significantly lower than those described above for SFO neurons. It would seem likely that such differences simply result from the use of sharp electrode recording techniques (OVLT), compared to whole cell patch (SFO), although confirmation of this conclusion will require further experiments. In all the cells recorded, the current–voltage plots reveal an outward rectification at potentials positive to 260 mV, when the cell is held at the resting membrane potential. If, however, the cell is held at potentials negative to 270 mV, depolarizing pulses elicit low threshold spikes (LTS) often accompanied by a brief burst of action potentials. As a consequence of this LTS activation, a strong inward rectification is observed in cells held at hyperpolarized potentials.

Circulating Factors and the Activity of OVLT Neurons

Angiotensin The effects of ANG on OVLT neurons have also been investigated, although the early work suffers from an inability to differentiate whether the cells under investigation were actually located within the borders of OVLT or whether they were from the larger, less distinct AV3V region. With this caveat in mind, such recordings do demonstrate that ANG has predominantly excitatory effects on OVLT/AV3V neurons both in vivo and in tissue slices (81, 107, 110). The effects are the result of direct ANG actions at its receptor since they can be blocked by administration of the ANG antagonist, saralasin (81, 110, 173). The lack of certainty with regard to the location of the cells is a major problem since the MnPO, which is also in the AV3V region, is similarly rich in ANG receptors. Unlike the OVLT, though, this region is protected by the BBB, and its likely source of ANG is either release at synapses or from the cerebral ventricles. More detailed, recent work has further supported the conclusion that ANG exerts excitatory actions on OVLT neurons, although other studies suggest that varying populations of these cells are influenced. A comparative study examining the responses of OVLT neurons in SHR versus WKY rats suggests that ANG evokes responses at lower doses in the SHR (107). Furthermore, the time required for the response to return to baseline after a brief ANG application was considerably longer in SHR, suggesting an enhanced efficacy of ANG

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receptors in SHR. However, the potential physiological significance of such observations has yet to be addressed. OVLT neurons have also been implicated in a number of other homeostatic mechanisms. They respond to increases in temperature by increasing their firing rate (99). This thermosensitivity persists in Ca21 free solution, implying that these cells are intrinsically capable of sensing changes in temperature. PGE2 , which is thought to mediate the febrile actions of pyrogens such as interleukin-1, also alters the discharge of the majority of OVLT neurons tested. It inhibits 65% of warm sensitive neurons and equally excites or inhibits thermally insensitive neurons (99). On the basis of this and other studies, it has been suggested that the OVLT may be the CNS site through which pyrogens act on the CNS to induce the febrile response (5, 79). The OVLT may also play a role in a negative feedback loop, regulating the secretion of LH. A study by Phillips and colleagues shows that GnRH has an inhibitory effect on OVLT neurons (36). This finding, however, has been disputed by others who suggest that the primary effect of GnRH on OVLT neurons is excitatory (133).

Osmotic Changes Intracellular recordings from OVLT neurons in a slice have shown that hypertonic artificial cerebrospinal fluid (aCSF) elicits a reversible depolarization in these cells (108), although whether such actions represent direct actions on these cells remain to be examined. Recent experiments from acutely dissociated OVLT neurons have begun to address this question by demonstrating that an increase in osmolarity results in a reversible depolarization which is accompanied by a decrease in input resistance, again suggesting the activation of a nonspecific cation channel (127). Once again, the FOS studies provide perhaps the best indication of the importance of the OVLT as a sensor of osmotic changes. Following hypertonic saline infusion, there is a dramatic increase in FOS expression in PVN, SON, and throughout the lamina terminalis (112). When combined with retrograde labeling, it is evident that a high percentage of the fibers which terminate on activated SON cells originate from cell bodies in OVLT (111).

OVLT Efferent Projections Involved in Neurosecretory Control

Almost all the work examining OVLT efferents has focused on its connections with SON. Electrical stimulation of OVLT neurons induces large (5–10 mV) bicuculline sensitive IPSPs in the majority of SON neurons (175). Blockade of these IPSPs reveals underlying fast EPSPs which are mediated by non-NMDA receptors and slower, NMDA-mediated EPSPs. Interestingly, the EPSP’s were only observed in vasopressinergic neurons (175). Hyperosmotic stimulation of the OVLT also increases EPSP frequency in SON, an effect which is accompa-

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nied by a reversible increase in neuronal discharge (126). Similarly, peptides such as ANP have been shown to excite SON neurons as a consequence of their actions at OVLT (125).

AREA POSTREMA

The AP is the most caudal of the CVOs, lying on the dorsal surface of the medulla immediately adjacent to the NTS. In rodents and lagomorphs it is a midline structure located at the obex of the fourth ventricle (172). In contrast, the AP of mammals such as humans and monkeys is a bilateral structure which protrudes into the lumen of the fourth ventricle (105). Classically, the primary function attributed to the AP was as the chemosensitive trigger zone in the emetic reflex (10, 11, 19, 103). More recently, functional roles for the AP in the control of cardiovascular regulation, food intake, cerebrospinal fluid regulation, and metabolism (8) have been demonstrated. The AP receives afferent input from, and sends extensive efferent projections to, autonomic control centers in the medulla, pons, and forebrain (162). Anterograde and retrograde tracing studies have demonstrated major efferent projections from the AP to NTS (137, 162) and lateral parabrachial nucleus (PBN) of the pons (22, 137, 162). There are also minor projections to dorsal motor nucleus of the vagus (DMV), dorsal and dorsolateral tegmental nuclei, and A1 area of the nucleus ambiguus (A1-NA) (137, 162). Afferent projections to the AP from the PVN of the hypothalamus, the PBN, the NTS (162), and the vagus nerve (26) have been morphologically characterized. AP afferents from the glossopharyngeal (25), carotid sinus (29), and aortic depressor (77) nerves have also been reported in the cat. These connections provide the anatomic framework through which the AP exerts its influence over the cardiovascular system. In addition, AP contains high densities of different groups of peptidergic receptors (101, 123). These specialized features provide the structural framework through which circulating substances which cannot cross the BBB may directly influence CNS neurons in this region.

Intrinsic Properties of AP Neurons

There is, to date, minimal direct evidence describing the specific properties of the AP neuronal membrane. This lack of information may be attributed primarily to the difficulties associated with obtaining intracellular recordings (either sharp electrode or whole cell patch) from small cells, especially when the cell of interest is located in a highly vascular region, rich in glial cells (23, 105). We would anticipate, however, that the dissociated cell techniques which we have recently developed for whole cell recording from SFO neurons (40), and which have been applied to calcium imaging studies from AP neurons (67), will be equally applicable to the study of membrane properties of AP neurons. Such technical developments will obviously be of vital importance to the develop-

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ment of an understanding of the specific mechanisms whereby AP cells can collect, integrate, and transduce vital information regarding the current autonomic status of the organism. Extracellular single-unit recording studies both in vivo and in vitro have, however, provided basic information regarding the overall function of AP neurons. The development of in vitro slice preparations of the rat and rabbit medulla (14, 16, 96), in which extracellular recordings can be routinely obtained from AP neurons, has demonstrated that the majority of these cells exhibit relatively low levels of spontaneous activity (, 1 Hz). Similar in vivo extracellular recordings have been obtained from the AP of pentobarbitol anesthetized animals (116–118, 142). It should be recognized, however, that such recordings (both in vivo and in vitro) may represent the properties of only a select population of AP neurons, i.e., those large enough to be recorded with the electrode being utilized and, perhaps more importantly, those exhibiting spontaneous activity. These technical considerations suggest that, if anything, the estimates of spontaneous activity from extracellular recordings may be too high. In addition, they emphasize the potential of recently developed techniques such as whole cell patch clamp recordings from dissociated neurons to make major contributions to the understanding of the physiology of AP.

Circulating Factors and the Activity of AP Neurons

The presence within the AP of receptors for different peptides may be interpreted as evidence indicating potential functions for such substances in controlling the activity of single neurons within this structure. Thus the reported high densities of binding sites for ANG (57, 102), VP (123), ET (76), ANP (3), cholecystokinin (CCK) (83), nerve growth factor (71), calcitonin gene-related peptide (CGRP (159), and amylin (135) indicate potential physiological roles for these peptides in controlling the function of AP neurons. It should, however, be emphasized at this point that apart from studies demonstrating functional roles for ANG and VP in central cardiovascular control (20, 50, 52, 95, 158, 167), the functional significance of the presence of high densities of other peptide receptors within AP is yet to be determined.

Angiotensin In addition to receptor localization studies, there is a considerable literature demonstrating significant cardiovascular actions of ANG within the AP. The iontophoretic studies of Carpenter et al. (17, 18) were the first to provide clear electrophysiological evidence that local administration of ANG influenced the activity of AP neurons in the dog. In the late 1980s, we were specifically interested in determining whether systemically administered ANG could, in fact, access AP neurons (as one would expect in view of the fenestrated

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FIG. 4. The effects of ANG on the activity of single AP neurons in vivo and in vitro. (A) Ratemeter recording (5 s/bin) from an AP neuron (lower histogram), and simultaneous mean arterial pressure recording (upper line) illustrating the excitatory effects of systemically administered ANG (arrow indicates time of administration) on this cell. (B) Shows a similar ratemeter recording obtained from an AP neuron in vitro demonstrating dose-dependent excitatory effects of ANG (time of bath administration indicated by solid bars).

capillaries in AP). We attempted to answer this question using extracellular single-unit recording techniques to examine the effects of intravenous ANG on the activity of these cells. These experiments showed excitatory actions of ANG on over 50% of the AP neurons from which we recorded (118), as illustrated in Fig. 4A. A further important finding emerged from the essential controls for these experiments in which we attempted to confirm that these actions of systemic ANG were direct effects of the peptide, rather than being a secondary consequence of the increase in blood pressure which occurs in response to administration of ANG. Such data were obtained by testing the same AP neurons with both ANG and a second similarly hypertensive agent to determine whether excitatory effects similar to those induced by ANG were also observed in response to increases in blood pressure. These experiments showed that while approximately half of the ANG-sensitive AP neurons were specifically responsive to ANG. The remaining cells influenced by ANG were also activated by methoxamine (118). In addition to confirming the ability of AP

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neurons to respond to circulating ANG, these data provided the first functional demonstration of potential baroreceptor input to these cells. Similar recent data showing that either systemic ANG or baroreceptor activation cause increased expression of c-fos in AP neurons (85, 100) are in accord with these electrophysiological findings. A second interpretation of the data which cannot be ruled out is that these ‘‘blood pressure-sensitive’’ neurons are simply responsive to both ANG and adrenergic agonists. Additional extracellular recordings in baroreceptor denervated animals would permit this question to be answered, but are not technically feasible in view of the instability associated with the increased blood pressure variability observed in denervated animals. Direct iontophoretic application of a-adrenergic antagonists onto AP neurons during intravenous agonist administration would answer this question. In the meantime the observation that single AP neurons may be responsive to circulating ANG and also receive direct primary afferent input from the baroreceptors further highlights the potential integrative importance of these cells. Significant recent attention has been directed toward understanding the specific cellular mechanisms through which ANG influences AP neurons. Information has been derived from two primary approaches: (1) extracellular recordings in medullary slice preparations (14, 16, 96) and (2) calcium imaging studies in dissociated AP neurons (67). Early in vitro recordings from AP neurons suggested that although a small population of these cells was responsive to certain cholinergic drugs, they were unaffected by low concentrations of ANG (1029 M ) (14). We have recently completed a more extensive analysis of the effects of bath administration of ANG on a population of 133 AP neurons and, as shown in Fig. 4B, have observed that approximately 40% of these cells were activated by ANG at doses of 1026 to 1029 M (144). In addition, we have been able to utilize the in vitro approach to determine that such effects of ANG result from direct actions of the peptide on AP neurons, as such excitatory actions are maintained in low Ca21 aCSF solutions which block synaptic transmission (Fig. 5). The increased stability of recordings obtained from this preparation (compared to in vivo recordings), combined with the ability to modify the slice perfusate rapidly, has allowed us also to demonstrate that effects of ANG on AP neurons are abolished by bath perfusion with the AT1 receptor antagonist, losartan (144). Although these studies confirm that ANG exerts direct actions on AP neurons mediated through AT1 receptors, they provide no direct information about the intracellular events mediating such actions. Recent Ca21 imaging studies provide some promise in this particular area (67). Using optical probes to measure real time changes in Ca21 concentrations of neonatal AP/medial NTS neurons maintained in tissue culture, Hay et al. (67) have demonstrated that intracellular Ca21 is increased in response to ANG administration and that such effects are abolished by losartan treatment. This preparation provides a potentially powerful tool with which to investigate the intracellular mechanisms underlying ANG actions on AP neurons using a combination of imaging and patch clamp techniques.

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FIG. 5. These two ratemeter records obtained from the same AP neuron illustrate excitatory effects of bath administration of ANG (time of administration indicated by hatched bars) in both normal and low Ca21 aCSF. Note that the effects of ANG are maintained in the latter medium, indicating that such effects are not dependent on maintained synaptic transmission in the slice and are thus most likely the result of direct actions on the neuron from which the recording was obtained.

Vasopressin Vasopressin receptors have been localized within the AP (123), while direct administration of VP into this CVO has been reported to increase blood pressure (95). In addition data derived from a number of different experimental approaches have clearly identified the AP as the essential CNS structure at which circulating VP acts to elicit its established effects on the baroreceptor reflex (1, 28, 63, 120, 160, 161). Together this information suggests significant roles for VP in controlling the activity of AP neurons. There are, however, relatively few studies which have attempted to evaluate directly the effects of VP on AP neurons. Carpenter et al. (17) were the first to report that iontophoretic application of VP elicited excitatory effects on quiescent AP neurons in the dog. We utilized our in vivo rat preparation for recording from AP neurons to investigate whether systemically administered VP exerted similar excitatory actions on this population of cells. We were initially gratified to find a large

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population of AP cells (45.8%) which responded to VP with decreases in activity (142), observations which led to the logical conclusion that these were the same population of cells which we had previously observed to be excited by ANG. The opposite actions of the peptides ANG and VP on these cells were interpreted as explaining how these two peptides exerted divergent effects on baroreflex control. However, further evaluation of these data revealed that 38% of AP neurons were activated by systemic VP and that if all cells influenced by circulating VP were considered, more than 80% were also influenced by changes in blood pressure. These data emphasize the major difficulties associated with interpretation of data derived from such in vivo experiments in isolation. We therefore diverted considerable attention to the development of our in vitro slice preparation so that we could definitively examine the direct actions of VP on AP neurons. Using this experimental approach, we were able to demonstrate that 63% of AP neurons were activated by bath administration of VP while only 6% were inhibited (96). More importantly, we were able to show that in all cases, excitatory actions of VP on AP neurons were maintained following blockade of synaptic transmission (achieved by perfusion of slices with aCSF in which Ca21 is replaced by Mg11 ), demonstrating these effects to be due to direct actions on the recorded neuron (96). We have used this slice preparation to verify that such effects of VP on AP neurons are mediated by V1 receptors and are dose dependent (96) as shown in Fig. 6. The question thus arises as to how we incorporate the differing data obtained from in vivo and in vitro studies into an integrated hypothesis explaining the actions of VP within the AP. Preliminary indications as to the intracellular events underlying such actions of VP on AP neurons can again be derived from the recent calcium imaging studies of Hay et al. (67). They report that cultural neonatal AP/mNTS neurons respond to VP with increases in intracellular calcium. Such changes in calcium could represent either primary effects of VP or be simply secondary to the presumed increase in action potentials occurring in these neurons in response to this peptide. Combined whole cell patch recordings and calcium imaging from the same AP neurons provide the technology through which we may soon be able to address the specific membrane events underlying these actions of VP on neurons in the AP.

Other Peptides Although the current literature provides persuasive arguments for physiological actions of ANG and VP in the AP, there are also data suggesting potential roles for additional circulating factors in controlling the activity of AP neurons. Microinjection of ET into AP has been shown to induce significant cardiovascular changes (48), while systemic ET increases the activity of AP neurons recorded in vivo (49). CCK receptors have also been identified in AP (176), and c-fos studies demonstrate that systemic CCK results in c-fos activation of AP neurons (54, 128), a small proportion of which remains following removal of vagal inputs (54). These data support the hypothesis of physiologically relevant

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FIG. 6. In vitro recordings from AP neurons illustrating effects of bath administration of VP. (A) Ratemeter recordings (5 s/bin) showing dose-dependent effects of VP (administration time indicated by bars) on a single AP neuron. (B) Histogram presenting the mean population effects of VP on AP neurons tested with doses of VP ranging from 1026 to 1028 M. (C) Continuous ratemeter (10 s/bin) recording from an AP neuron demonstrating effects of bath administration of VP (indicated by solid bar) and the ability of bath administration of a V1 receptor antagonist (indicated by hatched bar) to abolish this effect.

actions of CCK in AP. Our own recent in vitro extracellular recordings from AP neurons demonstrating excitatory actions of CCK on these neurons (145) are in agreement with this conclusion. It should be emphasized that the stability problems associated with recording in AP in vivo have, for the most part, precluded evaluation of single neuron responses to all of these peptides. We have recently begun in vitro studies designed to examine the sensitivity of single AP neurons to multiple peptides with the express goal of attempting to determine the specific peptidergic interests of separate populations of AP neurons. Initial data suggest that within AP there may exist specific groups of cells responsive to ANG, to ANG and VP, to VP, as well as to none of these peptides.

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In addition to the functional data described above, there is clear anatomical evidence supporting the view that AP neurons receive baroreceptor afferent inputs through the aortic depressor nerve in the cat (78). A similar projection has not been identified in the rat (24). We have nonetheless examined the functional consequences of aortic depressor nerve stimulation (selective baroreceptor afferent activation) on the activity of AP neurons, and we found that approximately 70% of AP cells receive excitatory inputs from this nerve (117). Although this experimental approach permits the conclusion that AP neurons receive information from the baroreceptors, we cannot distinguish at this point whether such inputs are mono- or polysynaptic. The anatomical description of reciprocal projections between AP and 1-PBN led us to electrophysiological studies which initially were designed to permit recording from AP neurons antidromically identified as projecting to this region. Less than 5% of AP neurons we recorded from could be antidromically activated, suggesting a minimal AP to 1-PBN projection. However, such a conclusion does not correspond with the available anatomical evidence (137, 162). It is possible that in our experiments the single unit recording techniques may preferentially select neurons which do not project to 1-PBN (based, for example, on cell size). Alternatively, our 1-PBN stimulation may not result in activation of AP axonal and terminal elements. On the other hand our electrophysiological analysis of orhtodromic effects permitted analysis of the functional nature of 1-PBN afferent input to AP. Approximately 25% of AP neurons recorded showed responses to electrical stimulation in 1-PBN with equal numbers of cells demonstrating excitatory or inhibitory effects (116). Anatomical tracing studies have demonstrated that the PVN represents one of the primary sources of afferent neural input to the AP (162). The potential functional significance of such a connection may be twofold in that (1) it provides a direct link between hypothalamic autonomic control centers and the AP and (2) this connection provides a mechanism for bisynaptic communication between the SFO and AP. We have recently investigated this connection between the PVN and the AP using in vivo single-unit recording studies and have demonstrated that over 30% of AP neurons tested are activated by electrical activation of neurons in PVN (141). Stimulation in regions immediately adjacent to the PVN, in contrast, was without effect, confirming that the observed responses were not simply the result of activation of fibers of passage. The relatively short latency (mean 28.2 6 3.3 ms, n 5 29) (141) of these effects may be interpreted as indicative of their monosynaptic nature, although it must be emphasized that definitive conclusions regarding this issue cannot be drawn from such electrophysiological evidence in isolation. AP Efferent Projections Involved in Autonomic Control

AP neurons have been suggested to play important roles in autonomic control, presumably through their efferent projections to autonomic centers in

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FIG. 7. The effects of different peptides on AP neurons. The subsequent effects of activation (1 excitatory, 2 inhibitory) of these cells on the activity of neurons in primary projection sites of AP is also illustrated.

the pons, medulla, and spinal cord. A number of studies have examined the effects of electrical stimulation of AP neurons on blood pressure (46, 51, 55, 64, 70, 140, 146), and a clear conclusion of such studies is that activation of AP neurons affects the cardiovascular system. Correlation of studies describing the effects of activation of AP neurons on the cardiovascular system with electrophysiological studies examining the actions of both electrical and chemical activation of AP cells on the activity of neurons in both primary and secondary projections sites of these cells represents an essential step to understanding the neuronal mechanisms through which AP exerts control over the autonomic nervous system. Considerable recent progress has been made in this area, as summarized in Fig. 7 The electrophysiological technique of antidromic activation theoretically permits classification of AP neurons according to their projection site (e.g., NTS, 1-PBN, etc). Some years ago, we anticipated that use of this technique would permit much clearer definition of the roles of specific populations of AP neurons and, in fact, initiated studies to examine the functional properties of AP neurons which projected to 1-PBN (116). Unfortunately, this experimental approach fell well short of our expectations. Despite the convincing anatomical data describing the projection to this nucleus, we were unable to antidromically identify more than about 2% of AP neurons as projecting to this site (116). The

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most likely explanation for the inability to activate this pathway is that the diameter of axons of AP neurons reaching the 1-PBN is so small that exceptionally large currents are required to elicit antidromic spikes. Notwithstanding these technical limitations, antidromic identification of AP neurons according to projection site still offers the potential to evaluate the functional characteristics of populations of AP neurons which are at least anatomically homogenous. Such an approach may ultimately provide a more coordinated view of the functions of these separate populations of AP neurons. Anatomical studies have clearly defined the NTS as receiving significant afferent input from AP neurons. Hay and Bishop (66) have utilized an in vitro medullary slice preparation to examine both the functional and the neurochemical nature of this primary output pathway from the AP. They have demonstrated that electrical activation of AP neurons results in activation of 86% of NTS neurons which could be isolated using extracellular recording techniques (i.e., those neurons which were either spontaneously active or synaptically driven by AP input) (66). These excitatory responses of NTS neurons were also found to be abolished by treatment of slices with the a2 adrenergic antagonist yohimbine, but were unaffected by the a1 antagonist, prazosin, indicating that the effects are mediated by catecholaminergic neurons activating a2 adrenergic receptors. Although these studies clearly demonstrate the existence of a dominant excitatory projection from AP to NTS, they suffer from the standard limitation of all such electrical stimulation studies in that this experimental approach results in collective activation of anatomically, rather than functionally, distinct populations of neurons. This issue was cleverly addressed by a subsequent series of studies from Bishop’s group in which activation of functionally distinct subpopulations of AP neurons was achieved by direct pressure ejection of ANG or VP into the AP while recording from NTS neurons identified as receiving input from the solitary tract (16). The data from such functional studies further emphasizes their potential significance; VP administered into AP elicited primarily excitatory actions on both spontaneous and solitary tract stimulation-induced activity of NTS neurons, while ANG actions were primarily inhibitory (73). These data, when combined with the demonstration that both ANG and VP exert primarily excitatory actions on AP neurons, clearly indicate that these two peptides influence separate populations of AP neurons, the activation of which is responsible for opposing effects on NTS neurons. The difficulty with such an interpretation rests with the lack of demonstration of inhibitory connections from AP to NTS (66), or within AP from ANG responsive neurons to VP responsive neurons (144). Other targets of efferent outputs of the AP which have been investigated using electrophysiological techniques include the 1-PBN (discussed above), the rostral ventrolateral medulla (RVLM), and the renal sympathetic nerves. Two separate studies have shown that RVLM neurons functionally identified as receiving sympathetic input (activity patterns correlated with sympathetic nerve discharge or cardiac cycle) respond to electrical stimulation in AP with an initial excitatory response which is then followed by a period of inhibition

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(146, 171). Intriguingly, the inhibitory and depressor effects of AP stimulation were both abolished by iontophoretic application of the GABAA antagonist bicuculline to the medullary neurons, while the excitatory effects on RVLM neurons were unaffected, and small pressor responses to AP stimulation replaced the previously observed depressor effect (146). These findings indicate that, as in the case of NTS, AP apparently forms functionally heterogenous synaptic connections with neurons in the RVLM, supporting the view of functionally distinct populations of neurons in the AP. The specificity of such inputs from AP to the RVLM is also highlighted by the observation that neurons in this region with activity patterns correlated with respiratory rhythm were unaffected by AP stimulation (171). Since the first studies to evaluate the effects of activation of AP neurons on cardiovascular function, it has been clear that the activity patterns of these cells have pronounced effects on the integrated multiunit activity which can be recorded in the renal sympathetic nerve (64, 68). Such studies have been consistent in their demonstration of reduced multiunit activity in response to both chemical (69) and electrical (64) activation of AP neurons.

NEUROHYPOPHYSIS AND MEDIAN EMINENCE

Often forgotten and frequently ignored, the neurohypophysis and median eminence are CVOs which for the most part have been left to get on with their neurosecretory functions in private. The SFO, OVLT, and AP are all neuronal cell body-rich CVOs in which it is believed that the primary functional reason for the lack of the blood–brain barrier is that this feature permits circulating factors which do not normally cross this barrier to gain direct access to neurons within the CNS. In contrast the neurohypophysis and median eminence are CVOs consisting primarily of axon terminals, and it would appear that the most important function associated with the lack of the normal BBB in these regions is that this feature permits release of peptides produced by neurosecretory neurons from nerve terminals into the general and hypophysial–portal circulation. However, it is also important to recognize the potential significance of circulating factors acting on these regions to modulate the specific cellular processes through which these nerve terminals secrete their neurohormones into the circulation. Within the median eminence or neurohypophysis, many different factors have now been shown to have specific modulatory actions on the secretion of neurohormones into the circulation (6, 80, 119, 130, 163). Recent patch clamp recordings from neurosecretory terminals have begun to identify the specific cellular processes involved in such secretion. In addition to the demonstration of nonspecific calcium dependent cationic conductance (84), a number of different types of voltage activated calcium currents have now been identified in neurohypophysial terminals (53, 166). Clearly identification of such channels represents a significant step toward elucidation of the specific

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mechanisms through which chemical messengers may act at these two CVOs to modulate the secretory process.

CONCLUSIONS

The circumventricular organs can be separated into two distinct classes according to their neuronal content and proposed roles in blood brain communication. The median eminence and neurophyophysis consist primarily of axons terminals. Their practical significance is clearly tied to the essential neurosecretory roles of these terminals, and the factors released from them, in regulation of the endocrine system. The SFO, AP, and OVLT all contain large numbers of neuronal cell bodies, high densities of peptidergic receptors, and send efferent connections to important local autonomic control centers. Afferent neural connections to these CVOs are at best sparse, and it has thus been proposed that the primary afferent control of these groups of neurons is derived from circulating chemical messengers. The functional significance of these structures appears to lie in their ability to monitor the physiological state of the ‘‘milieu interieur,’’ especially as it relates to changes from which structures inside the blood–brain barrier are protected and through their efferent connections implement appropriate homeostatic responses. There is now an immense literature supporting essential roles for these CVOs in the integrated control of body fluid balance, through primary regulation of drinking, pituitary hormone secretion, and the cardiovascular system. However, there is evidence that these structures may also be involved in reproductive function (36, 88), control of gastrointestinal motility (7), and emesis (8, 10, 12). The similarities among SFP, AP, and OVLT are perhaps more striking than their differences. They are morphologically very similar, and all appear to receive relatively sparse neural input, while sending extensive neural efferents to local autonomic control centers. They all contain high densities of a variety of different peptidergic receptors and have all been implicated as both osmosensitive regions and CNS sensors of circulating ANG. Single unit recording studies indicate that the majority of neurons in these structures have low rates of spontaneous activity (, 1 Hz). In addition the past 10 years have seen significant advances in our understanding of the specific roles of a number of different circulating factors in controlling the activity of these neurons. The large proportion of neurons influenced by each of these substances points clearly to the conclusion that single units are responsive to more than a single peptide. Understanding the specific interactions between these factors which take place at single CVO neurons will provide data essential to the design of future models of the neuronal circuitry through which these structures influence autonomic output. The recent application of in vitro extracellular and whole cell patch recording techniques to the study of these neurons offers considerable promise in this particular area. Electrophysiological studies have also provided important information describing the functional connections through which neurons

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in the SFO, AP, and OVLT have the capacity to exert significant influence over a large variety of physiological control systems.

ACKNOWLEDGMENTS Supported by the MRC of Canada and the Heart and Stroke Foundation of Ontario. J.S.B. is the holder of a scholarship from the Heart and Stroke Foundation of Canada.

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