Synaptic relationship between somatostatin- and neurokinin-1 receptor-immunoreactive neurons in the pre-Bötzinger complex of rats

J. Neurochem. (2012) 122, 923-933. ABSTRACT: The pre-B?tzinger complex (pre-B?tC) in the ventrolateral medulla oblongata is critical for the generation of respiratory rhythm in mammals. Somatostatin (SST) and neurokinin 1 receptor (NK1R) immunoreactivity have been used as markers of the pre-B?tC. SST immunoreactivity almost completely overlaps with small fusiform NK1R-immunoreactive (ir) neurons, the presumed rhythmogenic neurons, but not with large multipolar NK1R-ir neurons. Understanding the neurochemical characteristics, especially the synaptic relationship of SST/NK1R-ir neurons within the pre-B?tC network is essential in providing cellular and structural bases for understanding their physiological significance. This work has not been documented so far. We found that SST immunoreactivity was highly expressed in terminals, somas, and primary dendrites in the pre-B?tC. Besides the small fusiform neurons, a small population of medium-sized NK1R-ir neurons also colocalized with SST. Large NK1R-ir neurons were not SST-ir, but received somatostatinergic inputs. SST-ir terminals were glutamatergic or GABAergic, and synapsed with NK1R-ir neurons. Most of synapses between them were of the symmetric type, indicating their inhibitory nature. Asymmetric synapses were evident between SST-ir terminals and NK1R-ir dendrites, strongly suggesting an excitatory innervation from the presumed rhythmogenic neurons as these neurons are glutamatergic. We speculate that SST-mediated excitatory and inhibitory synaptic transmission onto NK1R-ir rhythmogenic and follower neurons synchronizes their activity to contribute to respiratory rhythmogenesis and control.     

Neurokinin-1 receptor-expressing neurons in the ventral medulla are essential for normal central and peripheral chemoreception in the conscious rat.

Neurokinin-1 receptor immunoreactive (NK1R-ir) neurons and processes are widely distributed within the medulla, prominently at central chemoreceptor sites. Focal lesions of NK1R-ir neurons in the medullary raphe or the retrotrapezoid nucleus partially reduced the CO(2) response in conscious rats. We ask if NK1R-ir cells and processes over a wide region of the ventral medulla are essential for central and peripheral chemoreception by cisterna magna injection of SSP-SAP, a high-affinity version of substance P-saporin. After 22 days, NK1R-ir cell loss was -79% in the retrotrapezoid nucleus and -65% in the A5 region, which lie close to the ventral surface, and -38% in the medullary raphe and -49% in the pre-B?tzinger complex/rostral ventral respiratory group, which lie deeper. Dorsal chemoreceptor sites, the caudal nucleus tractus solitarius and the A6 region, were unaffected. At 8 and 22 days, these lesions produced 1) hypoventilation during air breathing in wakefulness ( approximately 8%) and in non-rapid eye movement (NREM) ( approximately 9%) and rapid eye movement ( approximately 14%) sleep, as measured over a 4-h period; 2) a substantially reduced ventilatory response to 7% CO(2) by 61% in wakefulness and 46-57% in NREM sleep; and 3) a decreased ventilatory response to 12% O(2) by 40% in wakefulness and 35% in NREM sleep at 8 days, with partial recovery by 22 days. NK1R-ir neurons in the ventral medulla are essential for normal central chemoreception, provide a drive to breathe, and modulate the peripheral chemoreceptor responses. These effects are not state dependent.     

Glutamatergic inputs to the CVLM independent of the NTS promote tonic inhibition of sympathetic vasomotor tone in rats

GABAergic neurons in the caudal ventrolateral medulla (CVLM) are driven by baroreceptor inputs relayed via the nucleus tractus solitarius (NTS), and they inhibit neurons in rostral ventrolateral medulla to reduce sympathetic nerve activity (SNA) and arterial pressure (AP). After arterial baroreceptor denervation or lesions of the NTS, inhibition of the CVLM continues to increase AP, suggesting additional inputs also tonically activate the CVLM. This study examined whether the NTS contributes to baroreceptor-independent drive to the CVLM and whether glutamate promotes baroreceptor- and NTS-independent activation of the CVLM to tonically reduce SNA. In addition, we evaluated whether altering central respiratory drive, a baroreceptor-independent regulator of CVLM neurons, influences glutamatergic inputs to the CVLM. Splanchnic SNA and AP were measured in chloralose-anesthetized, ventilated, paralyzed rats. The infusion of nitroprusside decreased AP below threshold for baroreceptor afferent firing (<50 mmHg) and increased SNA to 209+/-22% (P<0.05), but the subsequent inhibition of the NTS by microinjection of the GABA(A) agonist muscimol did not further increase SNA. In contrast, after inhibition of the NTS, blockade of glutamatergic inputs to CVLM by microinjection of kynurenate increased SNA (274+/-54%; P<0.05; n=7). In vagotomized rats with baroreceptors unloaded, inhibition of glutamatergic inputs to CVLM evoked a larger rise in SNA when central respiratory drive was increased (219+/-16% vs. 271+/-17%; n=5; P<0.05). These data suggest that baroreceptor inputs provide the major drive for the NTS-mediated excitation of the CVLM. Furthermore, glutamate tonically activates the CVLM to reduce SNA independent of the NTS, and this excitatory input appears to be affected by the strength of central respiratory drive.     

The sympathoinhibitory effects of systemic cholecystokinin are dependent on neurons in the caudal ventrolateral medulla in the rat

The gastrointestinal hormone CCK inhibits a subset of presympathetic neurons in the rostroventrolateral medulla (RVLM) that may be responsible for driving the sympathetic vasomotor outflow to the gastrointestinal circulation. We tested the hypothesis that the central neurocircuitry of this novel sympathoinhibitory reflex involves a relay in the caudal ventrolateral medullary (CVLM) depressor area. Blood pressure and greater splanchnic sympathetic nerve discharge (SSND) or lumbar sympathetic nerve discharge (LSND) were monitored in anesthetised, paralyzed male Sprague-Dawley rats. The effects of phenylephrine (PE, 10 microg/kg iv; baroreflex activation), phenylbiguanide (PBG, 10 microg/kg iv; von Bezold-Jarisch reflex) and CCK (4 or 8 microg/kg iv) on SSND or LSND, were tested before and after bilateral injection of 50-100 nl of the GABAA agonist muscimol (1.75 mM; n=6, SSND; n=7, LSND) or the excitatory amino acid antagonist kynurenate (55 mM; n=7, SSND) into the CVLM. PE and PBG elicited splanchnic and lumbar sympathoinhibitory responses that were abolished by bilateral muscimol or kynurenate injection into the CVLM. Similarly, the inhibitory effect of CCK on SSND was abolished after neuronal inhibition within the CVLM. In contrast, CCK-evoked lumbar sympathoexcitation was accentuated following bilateral CVLM inhibition. In control experiments (n=7), these agents were injected outside the CVLM and had no effect on splanchnic sympathoinhibitory responses to PE, PBG, and CCK. In conclusion, neurons in the CVLM are necessary for the splanchnic but not lumbar sympathetic vasomotor reflex response to CCK. This strengthens the view that subpopulations of RVLM neurons supply sympathetic vasomotor outflow to specific vascular territories.     

Sympathoexcitatory CVLM neurons mediate responses to caudal pressor area stimulation

Neurons in the caudal pressor area (CPA) are a source of tonic sympathoexcitation that is dependent on activation of cardiovascular sympathetic premotor neurons in the rostral ventrolateral medulla (RVLM). In the present study, we sought to clarify the mechanism through which CPA neurons elicit increases in RVLM neuronal discharge, vasoconstrictor sympathetic tone, and arterial pressure. In urethan-chloralose-anesthetized, paralyzed, and artificially ventilated rats, bilateral disinhibition of CPA with bicuculline (Bic) after bilateral disinhibition of caudal ventrolateral medulla (CVLM) caused increases in splanchnic sympathetic nerve activity (+277% control) and arterial pressure (+54 mmHg). Inhibition of CVLM neurons with muscimol abolished the pressor response to activation of CPA neurons, suggesting that neurons within CVLM mediate the excitatory responses from CPA. Disinhibition of CVLM and CPA with Bic enhanced the sympathoexcitatory responses to stimulation of CPA with DL-homocysteic acid, which were blocked by microinjections of kynurenic acid into CVLM. We conclude that the pathway from CPA to RVLM involves an obligatory glutamatergic activation of sympathoexcitatory neurons in the vicinity of CVLM.     

Sympathoinhibitory pathway from caudal midline medulla to RVLM is independent of baroreceptor reflex pathway

Glutamate stimulation of the caudal midline medulla (CMM) causes profound sympathoinhibition due to GABAergic inhibition of presympathetic neurons in the rostral ventrolateral medulla (RVLM). We investigated whether the sympathoinhibitory pathway from CMM to RVLM, like the central baroreceptor reflex pathway, includes a glutamatergic synapse in the caudal ventrolateral medulla (CVLM). In pentobarbital sodium-anesthetized rats, the RVLM on one side was inhibited by a muscimol microinjection. Then the response evoked by glutamate microinjections into the CMM or by baroreceptor stimulation was determined before and after 1) microinjection of the GABA receptor antagonist bicuculline into the RVLM on the other side or 2) microinjections of the glutamate receptor antagonist kynurenate bilaterally into the CVLM. Bicuculline in the RVLM greatly reduced both CMM- and baroreceptor-evoked sympathoinhibition. Compared with the effect of vehicle solution, kynurenate in the CVLM greatly reduced baroreceptor-evoked sympathoinhibition, whereas its effect on CMM-evoked sympathoinhibition was not different from that of the vehicle solution. These findings indicate that the output pathway from CMM sympathoinhibitory neurons, unlike the baroreceptor and other reflex sympathoinhibitory pathways, does not include a glutamatergic synapse in the CVLM.     

Decompensated hemorrhage activates serotonergic neurons in the subependymal parapyramidal region of the rat medulla

According to prior evidence opioid and serotonin release by lower brain stem neurons may contribute to hemorrhage-induced sympathoinhibition (HISI). Here we seek direct evidence for the activation of opioidergic, GABAergic, or serotonergic neurons by severe hemorrhage in the medulla oblongata. Blood was withdrawn from awake rats (40-50% total volume) causing hypotension and profound initial bradycardia. Other rats received the vasodilator hydralazine, causing tachycardia and hypotension. Neuronal activation was gauged by the presence of Fos-immunoreactive (ir) nuclei after 2 h. Serotonergic, enkephalinergic, and GABAergic neurons were identified by the presence of a diagnostic enzyme or mRNA. Hemorrhaged rats had 30% fewer non-GABAergic Fos-ir neurons in the rostral ventrolateral medulla (RVLM) than hydralazine-treated rats, but they had six times more Fos-ir neurons within the subependymal parapyramidal nucleus (SEPPN). Fos-labeled SEPPN neurons were serotonergic (40-60%), GABAergic (31%), enkephalinergic (15%), or had mixed phenotypes. The data suggest that a reduced sympathoexcitatory drive from RVLM may contribute to HISI. SEPPN neuronal activation may also contribute to HISI or could mediate defensive thermoregulatory mechanisms triggered by hemorrhage-induced hypothermia.     

Leptin action on GABAergic neurons prevents obesity and reduces inhibitory tone to POMC neurons

Leptin acts in the brain to prevent obesity. The underlying neurocircuitry responsible for this is poorly understood, in part because of incomplete knowledge regarding first-order, leptin-responsive neurons. To address this, we and others have been removing leptin receptors from candidate first-order neurons. While functionally relevant neurons have been identified, the observed effects have been small, suggesting that most first-order neurons remain unidentified. Here we take an alternative approach and test whether first-order neurons are inhibitory (GABAergic, VGAT?) or excitatory (glutamatergic, VGLUT2?). Remarkably, the vast majority of leptin's antiobesity effects are mediated by GABAergic neurons; glutamatergic neurons play only a minor role. Leptin, working directly on presynaptic GABAergic neurons, many of which appear not to express AgRP, reduces inhibitory tone to postsynaptic POMC neurons. As POMC neurons prevent obesity, their disinhibition by leptin action on presynaptic GABAergic neurons probably mediates, at least in part, leptin's antiobesity effects.     

Vav3 is involved in GABAergic axon guidance events important for the proper function of brainstem neurons controlling cardiovascular, respiratory, and renal parameters

Vav3 is a phosphorylation-dependent activator of Rho/Rac GTPases that has been implicated in hematopoietic, bone, cerebellar, and cardiovascular roles. Consistent with the latter function, Vav3-deficient mice develop hypertension, tachycardia, and renocardiovascular dysfunctions. The cause of those defects remains unknown as yet. Here, we show that Vav3 is expressed in GABAegic neurons of the ventrolateral medulla (VLM), a brainstem area that modulates respiratory rates and, via sympathetic efferents, a large number of physiological circuits controlling blood pressure. On Vav3 loss, GABAergic cells of the caudal VLM cannot innervate properly their postsynaptic targets in the rostral VLM, leading to reduced GABAergic transmission between these two areas. This results in an abnormal regulation of catecholamine blood levels and in improper control of blood pressure and respiration rates to GABAergic signals. By contrast, the reaction of the rostral VLM to excitatory signals is not impaired. Consistent with those observations, we also demonstrate that Vav3 plays important roles in axon branching and growth cone morphology in primary GABAergic cells. Our study discloses an essential and nonredundant role for this Vav family member in axon guidance events in brainstem neurons that control blood pressure and respiratory rates.     

Does angiotensin II have a significant tonic action on cardiovascular neurons in the rostral and caudal VLM?

The peptidic ANG II receptor antagonists [Sar(1),Ile(8)]ANG II (sarile) or [Sar(1),Thr(8)]ANG II (sarthran) are known to decrease arterial pressure and sympathetic activity when injected into the rostral part of the ventrolateral medulla (VLM). In anesthetized rabbits and rats, the profound depressor and sympathoinhibitory response after bilateral microinjections of sarile or sarthran into the rostral VLM was unchanged after prior selective blockade of angiotensin type 1 (AT(1)) and ANG-(1---7) receptors, although this abolished the effects of exogenous ANG II. Unlike the neuroinhibitory compounds muscimol or lignocaine, microinjections of sarile in the rostral VLM did not affect respiratory activity. Sarile or sarthran in the caudal VLM resulted in a large pressor and sympathoexcitatory response, which was also unaffected by prior blockade of AT(1) and ANG-(1---7) receptors. The results indicate that the peptidic ANG receptor antagonists profoundly inhibit the tonic activity of cardiovascular but not respiratory neurons in the VLM and that these effects are independent of ANG II or ANG-(1---7) receptors.     

Increased GABA(A) inhibition of the RVLM after hindlimb unloading in rats

Attenuated baroreflex-mediated increases in renal sympathetic nerve activity (RSNA) in hindlimb unloaded (HU) rats apparently are due to changes within the central nervous system. We hypothesized that GABA(A) receptor-mediated inhibition of the rostral ventrolateral medulla (RVLM) is increased after hindlimb unloading. Responses to bilateral microinjection of the GABA(A) antagonist (-)-bicuculline methiodide (BIC) into the RVLM were examined before and during caudal ventrolateral medulla (CVLM) inhibition in Inactin-anesthetized control and HU rats. Increases in mean arterial pressure (MAP), heart rate (HR), and RSNA in response to BIC in the RVLM were significantly enhanced in HU rats. Responses to bilateral CVLM blockade were not different. When remaining GABA(A) inhibition in the RVLM was blocked by BIC during CVLM inhibition, the additional increases in MAP and RSNA were significantly greater in HU rats. These data indicate that GABA(A) receptor-mediated inhibition of RVLM neurons is augmented after hindlimb unloading. Effects of input from the CVLM were unaltered. Thus, after cardiovascular deconditioning in rodents, the attenuated increase in sympathetic nerve activity in response to hypotension is associated with greater GABA(A) receptor-mediated inhibition of RVLM neurons originating at least in part from sources other than the CVLM.     

Sympathoinhibition from ventrolateral periaqueductal gray mediated by 5-HT(1A) receptors in the RVLM

The role of 5-hydroxytryptamine 1A (5-HT(1A)) receptors located in the rostral ventrolateral medulla (RVLM) in the mediation of a sympathoinhibitory and depressor response elicited from the ventrolateral periaqueductal gray (vlPAG) matter of the midbrain was examined in pentobarbital sodium-anesthetized rats. Activation of neurons in the vlPAG evoked a decrease in renal and lumbar sympathetic nerve activities and a decrease in arterial blood pressure. After microinjection of the specific 5-HT(1A)-receptor antagonist WAY-100635 into the pressor area of the RVLM, the vlPAG-evoked sympathoinhibition and hypotension was attenuated to control levels (7 of 15 animals) or converted into a sympathoexcitation and pressor response (8 of 15 animals). Baroreflex inhibition of sympathetic nerve activity was not impaired by microinjection of WAY into the sympathoexcitatory region of the RVLM. These data suggest that sympathoinhibition and hypotension elicited by activation of neurons in the vlPAG are mediated by 5-HT(1A) receptors in the RVLM.     

Attenuation of homeostatic responses to hypotension and glucoprivation after destruction of catecholaminergic rostral ventrolateral medulla neurons

This study determined the effect of destruction of rostral ventrolateral medulla (RVLM)-C1 cells on integrated sympathetic and hormonal responses to hypotension or glucoprivation. Injection of anti-dopamine beta-hydroxylase-saporin into the RVLM resulted in 29-99% depletion of RVLM-C1 neurons and approximately 60% reduction in the number of A5 neurons. As in our previous study in unanesthetized rats, resting mean arterial pressure (MAP) was reduced by approximately 10 mmHg in rats with >80% depletion of RVLM-C1 cells compared with control rats, although resting heart rate (HR) did not differ significantly. In the present study, resting plasma levels of norepinephrine (NE) did not differ significantly between control rats and rats with >80% depletion of RVLM-C1 cells, although there was a tendency for RVLM-C1 lesioned rats to have lower levels. Also consistent with our previous study, hydralazine (HDZ)-evoked hypotension resulted in smaller increases in HR and plasma levels of NE in rats with >80% depletion of RVLM-C1 cells compared with control rats. Furthermore, the elevated plasma levels of posterior pituitary hormones vasopressin and oxytocin evoked by HDZ were blunted in RVLM-C1 lesioned rats compared with control rats, even though MAP fell to lower levels in the lesioned rats. Plasma renin activity, plasma osmolality, and plasma protein concentrations did not differ between control rats and rats with >80% depletion of RVLM-C1 neurons. In response to systemic administration of 2-deoxyglucose, the circulating level of epinephrine and the resulting hyperglycemia were attenuated in rats with >80% depletion of RVLM-C1 cells compared with control rats. These results demonstrate that RVLM-C1 cells, in addition to playing a role in acute cardiovascular reflexes, play an important role in integrated sympathetic and hormonal responses to homeostatic challenges such as hypotension and glucoprivation.     

A Very Large Number of GABAergic Neurons Are Activated in the Tuberal Hypothalamus during Paradoxical (REM) Sleep Hypersomnia

We recently discovered, using Fos immunostaining, that the tuberal and mammillary hypothalamus contain a massive population of neurons specifically activated during paradoxical sleep (PS) hypersomnia. We further showed that some of the activated neurons of the tuberal hypothalamus express the melanin concentrating hormone (MCH) neuropeptide and that icv injection of MCH induces a strong increase in PS quantity. However, the chemical nature of the majority of the neurons activated during PS had not been characterized. To determine whether these neurons are GABAergic, we combined in situ hybridization of GAD₆₇ mRNA with immunohistochemical detection of Fos in control, PS deprived and PS hypersomniac rats. We found that 74% of the very large population of Fos-labeled neurons located in the tuberal hypothalamus after PS hypersomnia were GAD-positive. We further demonstrated combining MCH immunohistochemistry and GAD₆₇ in situ hybridization that 85% of the MCH neurons were also GAD-positive. Finally, based on the number of Fos-ir/GAD⁺, Fos-ir/MCH⁺, and GAD⁺/MCH⁺ double-labeled neurons counted from three sets of double-staining, we uncovered that around 80% of the large number of the Fos-ir/GAD⁺ neurons located in the tuberal hypothalamus after PS hypersomnia do not contain MCH. Based on these and previous results, we propose that the non-MCH Fos/GABAergic neuronal population could be involved in PS induction and maintenance while the Fos/MCH/GABAergic neurons could be involved in the homeostatic regulation of PS. Further investigations will be needed to corroborate this original hypothesis.     

Development and evolution of cerebellar neural circuits

The cerebellum controls smooth and skillful movements and it is also involved in higher cognitive and emotional functions. The cerebellum is derived from the dorsal part of the anterior hindbrain and contains two groups of cerebellar neurons: glutamatergic and gamma-aminobutyric acid (GABA)ergic neurons. Purkinje cells are GABAergic and granule cells are glutamatergic. Granule and Purkinje cells receive input from outside of the cerebellum from mossy and climbing fibers. Genetic analysis of mice and zebrafish has revealed genetic cascades that control the development of the cerebellum and cerebellar neural circuits. During early neurogenesis, rostrocaudal patterning by intrinsic and extrinsic factors, such as Otx2, Gbx2 and Fgf8, plays an important role in the positioning and formation of the cerebellar primordium. The cerebellar glutamatergic neurons are derived from progenitors in the cerebellar rhombic lip, which express the proneural gene Atoh1. The GABAergic neurons are derived from progenitors in the ventricular zone, which express the proneural gene Ptf1a. The mossy and climbing fiber neurons originate from progenitors in the hindbrain rhombic lip that express Atoh1 or Ptf1a. Purkinje cells exhibit mediolateral compartmentalization determined on the birthdate of Purkinje cells, and linked to the precise neural circuitry formation. Recent studies have shown that anatomy and development of the cerebellum is conserved between mammals and bony fish (teleost species). In this review, we describe the development of cerebellar neurons and neural circuitry, and discuss their evolution by comparing developmental processes of mammalian and teleost cerebellum.     

Evidence That Adrenergic Ventrolateral Medullary Cells Are Activated whereas Precerebellar Lateral Reticular Nucleus Neurons Are Suppressed during REM Sleep

Rapid eye movement sleep (REMS) is generated in the brainstem by a distributed network of neurochemically distinct neurons. In the pons, the main subtypes are cholinergic and glutamatergic REMS-on cells and aminergic REMS-off cells. Pontine REMS-on cells send axons to the ventrolateral medulla (VLM), but little is known about REMS-related activity of VLM cells. In urethane-anesthetized rats, dorsomedial pontine injections of carbachol trigger REMS-like episodes that include cortical and hippocampal activation and suppression of motoneuronal activity; the episodes last 4–8 min and can be elicited repeatedly. We used this model to determine whether VLM catecholaminergic cells are silenced during REMS, as is typical of most aminergic neurons studied to date, and to investigate other REMS-related cells in this region. In 18 anesthetized, paralyzed and artificially ventilated rats, we obtained extracellular recordings from VLM cells when REMS-like episodes were elicited by pontine carbachol injections (10 mM, 10 nl). One major group were the cells that were activated during the episodes (n = 10). Their baseline firing rate of 3.7±2.1 (SD) Hz increased to 9.7±2.1 Hz. Most were found in the adrenergic C1 region and at sites located less than 50 µm from dopamine β-hydroxylase-positive (DBH⁺) neurons. Another major group were the silenced or suppressed cells (n = 35). Most were localized in the lateral reticular nucleus (LRN) and distantly from any DBH⁺ cells. Their baseline firing rates were 6.8±4.4 Hz and 15.8±7.1 Hz, respectively, with the activity of the latter reduced to 7.4±3.8 Hz. We conclude that, in contrast to the pontine noradrenergic cells that are silenced during REMS, medullary adrenergic C1 neurons, many of which drive the sympathetic output, are activated. Our data also show that afferent input transmitted to the cerebellum through the LRN is attenuated during REMS. This may distort the spatial representation of body position during REMS.     

The nNos/cGMP mediation of the depressor response to NMDA receptor stimulation in the caudal ventrolateral medulla

Using in vivo voltammetry to directly measure extracellular nitric oxide (NO) levels, our previous studies suggested that the neuronal NO synthase (nNOS) and cyclic guanosine monophosphate (cGMP) signal transducing systems are involved in the cardiovascular responses elicited by activation of N-methyl-D-aspartate (NMDA) receptors in the rostral ventrolateral medulla. In this study, we examined if the depressor responses elicited by activation of NMDA receptors in the caudal ventrolateral medulla (CVLM) also depend on the actions of nNOS and soluble guanylyl cyclase. In anesthetized cats, microinjection of NMDA into the CVLM produced hypotension and bradycardia associated with NO formation. These NMDA-induced responses were attenuated by prior injections of 2-amino-5-phosphonopentanoate (a NMDA receptor competitive antagonist), 7-nitroindazole (a nNOS inhibitor) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase). These findings suggest that NO is also involved in the NMDA-induced depressor responses of the CVLM.     

Functional Organization of Brain Pathways Subserving the Baroreceptor Reflex: Studies in Conscious Animals Using Immediate Early Gene Expression

1. This paper reviews studies carried out in our laboratory in which we have used the c-fos functional mapping method, in combination with other methods, to determine the functional organization of central baroreceptor pathways as they operate in the conscious rabbit.2. First, we showed that periods of induced hypertension or hypotension each result in a specific and reproducible pattern of activation of neurons in the brainstem and forebrain. In particular, hypotension (but not hypertension) results in the activation of catecholamine neurons in the medulla and pons and vasopressin-synthesizing neurons in the hypothalamus.3. The activation of medullary cell groups in response to induced hypertension or hypotension in the conscious rabbit is almost entirely dependent on inputs from arterial baroreceptors, while the activation of hypothalamic vasopressin-synthesising neurons in response to hypotension is largely dependent on baroreceptors, although an increase in circulating angiotensin also appears to contribute.4. Discrete groups of neurons in the rostral ventrolateral medulla (RVLM) and A5 area in the pons are the major groups of spinally projecting neurons activated by baroreceptor unloading. In contrast, spinally projecting neurons in the paraventricular nucleus in the hypothalamus appear to be largely unaffected by baroreceptor signals.5. Direct afferent inputs to RVLM neurons in response to increases or decreases in arterial pressure originate primarily from other medullary nuclei, particularly neurons located in the caudal and intermediate levels of the ventrolateral medulla (CVLM and IVLM), as well as in the nucleus tractus solitarius (NTS).6. There is also a direct projection from barosensory neurons in the NTS to the CVLM/IVLM region, which is activated by baroreceptor inputs.7. Collectively, the results of our studies in conscious animals indicate that baroreceptor signals reach all levels of the brain. With regard to the baroreceptor reflex control of sympathetic activity, our studies are consistent with previous studies in anesthetized animals, but in addition reveal other previously unrecognized pathways that also contribute to this reflex regulation.     

Circadian control of the daily plasma glucose rhythm: an interplay of GABA and glutamate

The mammalian biological clock, located in the hypothalamic suprachiasmatic nuclei (SCN), imposes its temporal structure on the organism via neural and endocrine outputs. To further investigate SCN control of the autonomic nervous system we focused in the present study on the daily rhythm in plasma glucose concentrations. The hypothalamic paraventricular nucleus (PVN) is an important target area of biological clock output and harbors the pre-autonomic neurons that control peripheral sympathetic and parasympathetic activity. Using local administration of GABA and glutamate receptor (ant)agonists in the PVN at different times of the light/dark-cycle we investigated whether daily changes in the activity of autonomic nervous system contribute to the control of plasma glucose and plasma insulin concentrations. Activation of neuronal activity in the PVN of non-feeding animals, either by administering a glutamatergic agonist or a GABAergic antagonist, induced hyperglycemia. The effect of the GABA-antagonist was time dependent, causing increased plasma glucose concentrations only when administered during the light period. The absence of a hyperglycemic effect of the GABA-antagonist in SCN-ablated animals provided further evidence for a daily change in GABAergic input from the SCN to the PVN. On the other hand, feeding-induced plasma glucose and insulin responses were suppressed by inhibition of PVN neuronal activity only during the dark period. These results indicate that the pre-autonomic neurons in the PVN are controlled by an interplay of inhibitory and excitatory inputs. Liver-dedicated sympathetic pre-autonomic neurons (responsible for hepatic glucose production) and pancreas-dedicated pre-autonomic parasympathetic neurons (responsible for insulin release) are controlled by inhibitory GABAergic contacts that are mainly active during the light period. Both sympathetic and parasympathetic pre-autonomic PVN neurons also receive excitatory inputs, either from the biological clock (sympathetic pre-autonomic neurons) or from non-clock areas (para-sympathetic pre-autonomic neurons), but the timing information is mainly provided by the GABAergic outputs of the biological clock.     

Role of ventrolateral medulla in generating the 10-Hz rhythm in sympathetic nerve discharge

We recorded changes in right inferior cardiac and either left inferior cardiac or left vertebral sympathetic nerve discharge (SND) produced by unilateral microinjections of GABA-A and excitatory amino acid (EAA) receptor antagonists into the ventrolateral medulla (VLM) of urethane-anesthetized, baroreceptor-denervated cats. Unilateral microinjections of GABA-A receptor antagonists, SR-95531 or bicuculline, into single tracks in VLM anywhere between 1 and 5 mm rostral to the obex eliminated or markedly reduced 10-Hz power in SND on both sides of the body. Low-frequency components (<6 Hz) of SND were unaffected. Complete blockade of the 10-Hz rhythm occurred with a dose of SR-95531 as low as 6.25 pmol in a 50-nl volume. Unilateral microinjections of the nonselective EAA receptor antagonist, kynurenate (KYN; 7.5 nmol), into the caudal or rostral VLM significantly reduced, but did not eliminate, 10-Hz SND ipsilateral to the injection sites, while 10-Hz SND contralateral to the injection sites was not significantly changed. These observations suggest that 1) GABAergic transmission in VLM is critical for generation of the 10-Hz rhythm, 2) the caudal and rostral portions of VLM act together to generate the 10-Hz rhythm, and 3) 10-Hz rhythm generation depends, at least in part, on tonic or phasic excitatory drive to GABAergic interneurons in caudal VLM and presympathetic neurons in rostral VLM. The data also suggest that pathways interconnecting the two halves of the brain stem play an important role in promoting 10-Hz rhythm generation.