Stimulation of the rat subthalamic nucleus is neuroprotective following significant nigral dopamine neuron loss.

Deep brain stimulation of the subthalamic nucleus (STN-DBS) is efficacious in treating the motor symptoms of Parkinson's disease (PD). However, the impact of STN-DBS on the progression of PD is unknown. Previous preclinical studies have demonstrated that STN-DBS can attenuate the degeneration of a relatively intact nigrostriatal system from dopamine (DA)-depleting neurotoxins. The present study examined whether STN-DBS can provide neuroprotection in the face of prior significant nigral DA neuron loss similar to PD patients at the time of diagnosis. STN-DBS between 2 and 4 weeks after intrastriatal 6-hydroxydopamine (6-OHDA) provided significant sparing of DA neurons in the SN of rats. This effect was not due to inadvertent lesioning of the STN and was dependent upon proper electrode placement. Since STN-DBS appears to have significant neuroprotective properties, initiation of STN-DBS earlier in the course of PD may provide added neuroprotective benefits in addition to its ability to provide symptomatic relief.

The effects of diphenhydramine and SR142948A on periaqueductal gray neurons and on the interactions between the medial preoptic nucleus and the periaqueductal gray.

The midbrain periaqueductal gray contains both neurotensin type-1 and type-2 receptors. Behavioral studies have shown that the analgesic effect of neurotensin is mediated through its interaction with the type-2 receptors. These receptors specifically bind the type-1 histamine antagonist, levocabastine. Recently, it has been shown that another histamine-1 antagonist, diphenhydramine, blocks the analgesic effect of neurotensin. In addition, it has been shown that a non-peptide neurotensin antagonist, SR142948A, binds to both types of neurotensin receptors and blocks the analgesic effect of exogenously applied neurotensin. Major afferents to the periaqueductal gray arise from the medial preoptic nucleus of the hypothalamus. This region contains neurotensinergic neurons, and the expression of neurotensin mRNA in this region increases following cold-water swim stress that leads to opioid-independent analgesia. The goal of this study was to determine whether the responses of periaqueductal gray neurons to stimulation of the medial preoptic nucleus are modified by local injection of diphenhydramine and SR142948A. Because the cellular basis of the effects of diphenhydramine on periaqueductal gray neurons had not been reported, we also examined the effects of diphenhydramine on the baseline-firing rate and synaptic transmission using in vivo and in vitro methods. The results of the in vitro studies indicate that diphenhydramine concentrations above 500 nM significantly reduce the baseline firing of the periaqueductal gray neurons without a significant effect on the frequency of postsynaptic potentials. At concentrations below 100 nM, diphenhydramine has little effect on the baseline-firing rate but partially blocks the response to neurotensin. The results of the in vivo studies showed similar effects of diphenhydramine. At high concentrations it inhibited periaqueductal gray neurons, but at low concentrations it had no effect on the baseline-firing rate and it blocked the response to neurotensin and to medial preoptic nucleus stimulation. Unlike diphenhydramine, SR142948A had virtually no effect on the baseline-firing rate but blocked the response to neurotensin and to stimulation of the medial preoptic nucleus. It is concluded that: (1) SR142948A, at a dose that completely blocks the effect of exogenously applied neurotensin on periaqueductal gray neurons, has little effect on their baseline-firing rates. (2) Because of its effect on the baseline-firing rate, only low doses of diphenhydramine can be used as an antagonist of the neurotensin analgesic effect. (3) Responses of periaqueductal gray neurons to medial preoptic nucleus stimulation is, in part, mediated by a neurotensinergic network within the periaqueductal gray.

Toward a rational understanding of migraine trigger factors.

The typical migraine patient is exposed to a myriad of migraine triggers on a daily basis. These triggers potentially can act at various sites within the cerebral vasculature and the central nervous system to promote the development of migraine headache. The challenge to the physician is in the identification and avoidance of migraine trigger factors within patients suffering from migraine headache. Only through a rational approach to migraine trigger factors can physicians develop an appropriate treatment strategy for migraine patients.

Cold water swim stress increases the expression of neurotensin mRNA in the lateral hypothalamus and medial preoptic regions of the rat brain.

Stress-induced analgesia is a well-documented phenomenon that occurs in all mammalian species. Forced cold water swim produces a type of stress-induced analgesia that is independent of mu opioid receptors. The neuropeptide neurotensin (NT) has been implicated in mu opioid-independent analgesia (MOIA), but the circuitry of this system is largely unknown. The medial preoptic area (MPO) and lateral hypothalamus (LH) are two regions that are known to modulate pain processing. These two regions also contain neurotensinergic projections to the periaqueductal gray, a region that has been shown to produce MOIA upon injection of NT. The goal of this study was to determine if cold water swim (CWS) stress, which produces MOIA, activates the NT-ergic systems in these two regions. In situ hybridization results indicate that CWS increases the level of NT mRNA within neurons in the MPO and LH, suggesting that these two regions are activated during this process.

Identification and transgenic analysis of a murine promoter that targets cholinergic neuron expression.

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using beta-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.

Synaptic effects of nitric oxide on enkephalinergic, GABAergic, and glutamatergic networks of the rat periaqueductal gray.

Previous studies have shown that the injection of nitric oxide (NO) donating compounds into the dorsal periaqueductal gray region of the midbrain (PAG) decreases mean arterial pressure (MAP), while the injection of NO synthase (NOS) inhibitors increases MAP. In this study we used both in-vivo and in-vitro preparations and examined the effect of a NO donor and a NOS inhibitor on MAP, membrane properties, and synaptic activities in PAG neurons. We found that: (1) Injection of the NO donor hydroxylamine (HA) into the dorsal PAG decreased MAP, while the injection of the neuronal NOS (nNOS) inhibitor, 1-(2-trifluoromethylphenyl) imidazole (TRIM) increased MAP. These responses were consistent and site-specific. (2) HA-evoked hypotensive responses were mediated by PAG neuronal activity, because they were blocked by pre-injection with gamma-amino-butyric acid (GABA). (3) HA consistently increased the rate of observable synaptic events while TRIM consistently decreased the rate of observable synaptic events. (4) Bicuculline (BIC) and naloxone (NAL) blocked HA-evoked increases in the rate of observable inhibitory synaptic events. (5) Perfusion with sodium nitroprusside (SNP) and illumination with bright light consistently elevated rates of observable synaptic events, and SNP-evoked increases of excitatory synaptic events were blocked by pretreatment with glutamic acid antagonists. (6) PAG-medullary projecting neurons exhibited similar response patterns. The results of this study suggest that: (1) NO production within the PAG is a major component of PAG-mediated cardiovascular responses. (2) The effects of NO may be mediated in part by increased presynaptic vesicular release of glutamic acid, GABA, and enkephalin.

The medial preoptic nucleus of the hypothalamus modulates activity of nitric oxide sensitive neurons in the midbrain periaqueductal gray.

Stimulation of the medial preoptic nucleus of the hypothalamus (MPO) has been shown to produce decreases in mean arterial pressure (MAP) by a pathway involving the periaqueductal gray region of the midbrain (PAG). Previous studies have shown that the injection of nitric oxide (NO) donating compounds into the dorsal PAG also decreases MAP, while the injection of nitric oxide synthase (NOS) inhibitors increases MAP. Collectively these studies suggest that the MPO elicited hypotensive response may involve NO production in PAG neurons. In this study, we investigated this hypothesis. We found that: (1) Bilateral injection of the NOS inhibitor 7-nitro indazole (7-NI) into the dorsolateral PAG cell columns produced elevations in MAP in a highly consistent and site specific fashion. (2) Microinjection of 7-NI in quantities that were too low to directly influence MAP blocked the MPO evoked hypotensive response in 9/11 cases. (3) While 41% of dorsal PAG neurons had baseline firing rates that were sensitive to 7-NI, 69% of PAG neuronal responses to MPO stimulation were blocked by 7-NI. (4) Inhibitory responses that were not blocked by 7-NI had significantly shorter latencies to onset in the presence of 7-NI. (5) PAG neurons that projected to the medulla exhibited similar electrophysiologic response patterns. Our results suggest the following: (1) The dorsolateral PAG contains a NO producing hypotensive network. (2) The MPO elicited hypotensive response may utilize this network. (3) Stimulation of the MPO elicits NO dependent responses from PAG neurons, some of which do project to medullary-cardiovascular control centers.

Annexin VI modulates Ca2+ and K+ conductances of spinal cord and dorsal root ganglion neurons.

Annexin VI is a member of a Ca(2+)-dependent phospholipid-binding protein family that participates in the transduction of the intracellular Ca2+ signal. We have identified annexin VI as one of the major annexins expressed differentially by sensory neurons of dorsal root ganglia (DRG) and by neurons of spinal cord (SC) of the rat and the mouse. This annexin shows a preferential localization at the plasma membrane of the soma and cellular processes, particularly in motoneurons of the SC. This finding suggests an active role of annexin VI in the Ca(2+)-dependent regulation of plasma membrane functions. To test this possibility, the neuronal function of annexin VI was evaluated by whole cell electrophysiology of mouse embryo SC and DRG neurons. An antibody was developed that has the property of neutralizing annexin VI-phospholipid interactions. The intracellular perfusion of individual neurons in culture, either from SC or DRG, with monospecific affinity-purified anti-annexin VI antibodies resulted in an increase in the magnitude of the K+ current and in an increase in the Ca2+ current in sensory neurons. Our results suggest that the endogenous annexin VI regulates the Ca2+ conductance, which indirectly modifies Ca(2+)-dependent ionic conductances in SC and DRG neurons.

Properties of a projection pathway from the medial preoptic nucleus to the midbrain periaqueductal gray of the rat and its role in the regulation of cardiovascular function.

In this study we examined (1) the effect of stimulation of the MPO on the firing activity of neurons in the PAG, (2) the role of glutamic acid in this interaction and, (3) whether reversible blockade of neuronal activity in the PAG by lidocaine can alter the effect of stimulation of the MPO on arterial blood pressure. Single pulse stimulation of the MPO produced a biphasic response in 2/32 cells and inhibited 3/32 cells. Train electrical stimulation excited 21/54 cells and inhibited 12/54 cells. The latencies to the onset of the excitatory and the inhibitory effects were not different, but the duration of the excitatory effect was slightly longer than that of the inhibitory effect. Chemical stimulation of the MPO excited 17/97 cells and inhibited 16/97 cells. The latency to onset of the excitatory response to stimulation of the MPO was longer but the duration was shorter than that of the inhibitory response. In 83% of the animals (29/35), stimulation of the MPO produced a decrease in mean arterial pressure (MAP). The duration of the response was 196.9 +/- 20.9 s and the average decrease in the MAP was 18.2 +/- 1.4 mmHg. Application of KYN blocked the excitatory response to stimulation of the MPO in 8/16 cells and the inhibitory response of 3/10 cells. Injection of lidocaine into the PAG by itself had no effect on the arterial blood pressure. However, in all animals (n = 10) lidocaine totally or significantly reduced the magnitude of the blood pressure change produced by stimulation of the MPO in a reversible manner. These studies electrophysiologically confirm a pathway between the MPO and the PAG that is, in part, under glutamatergic control. In addition, our results demonstrate that stimulation of the MPO produces a distinctive depressor effect that is mediated through the PAG.

The role of the basolateral nucleus of the amygdala in the pathway between the amygdala and the midbrain periaqueductal gray in the rat.

We electrophysiologically examined the connection between the basolateral nucleus of the amygdala (BLA) and the periaqueductal gray (PAG) and examined the role of the central nucleus of the amygdala (CNA) in this pathway. Train electrical stimulation of the BLA excited 21% (7/33) and inhibited 27% (9/33) of the cells recorded in the PAG. Chemical stimulation of the BLA excited 23% (13/56) and inhibited 16% (9/56) of the cells recorded in the PAG. Injection of lidocaine into the CNA by itself had no effect on PAG cells (n = 9) or on blood pressure but blocked the effect of BLA stimulation on PAG neurons in 78% of the cells recorded. It was concluded that: (1) PAG cells respond to BLA stimulation; (2) the majority of these cells are located in the dorsolateral and lateral columns of the PAG; and (3) the CNA modulates a majority of the activities of the BLA in the PAG.

Differential expression of annexins I-VI in the rat dorsal root ganglia and spinal cord.

The annexins are a family of Ca(2+)-dependent phospholipid-binding proteins. In the present study, the spatial expression patterns of annexins I-VI were evaluated in the rat dorsal root ganglia (DRG) and spinal cord (SC) by using indirect immunofluorescence. Annexin I is expressed in small sensory neurons of the DRG, by most neurons of the SC, and by ependymal cells lining the central canal. Annexin II is expressed by most sensory neurons of the DRG but is primarily expressed in the SC by glial cells. Annexin III is expressed by most sensory neurons, regardless of size, by endothelial cells lining the blood vessels, and by the perineurium. In the SC, annexin III is primarily expressed by astrocytes. In the DRG and the SC, annexin IV is primarily expressed by glial cells and at lower levels by neurons. In the DRG, annexin V is expressed in relatively high concentrations in small sensory neurons in contrast to the SC, where it is expressed mainly by ependymal cells and by small-diameter axons located in the superficial laminae of the dorsal horn areas. Annexin VI is differentially expressed by sensory neurons of the DRG, being more concentrated in small neurons. In the SC, annexin VI has the most striking distribution. It is concentrated subjacent to the plasma membrane of motor neurons and their processes. The differential localization pattern of annexins in cells of the SC and DRG could reflect their individual biological roles in Ca(2+)-signal transduction within the central nervous system.

Intact female rats are more susceptible to the development of tactile allodynia than ovariectomized female rats following partial sciatic nerve ligation (PSNL).

As previously reported, this laboratory has determined that female rats are more prone to develop tactile allodynia, an indicator of neuropathic pain, than male rats using the partial sciatic nerve ligation (PSNL) model. In order to further characterize this gender difference, the role of ovarian hormones in predisposing female rats to the development of tactile allodynia was investigated. In a double blind randomized trial, 12 intact and 12 ovariectomized (ovx) female rats underwent PSNL of the right rear leg. Sham 1 operated (exposure of the nerve without ligation; 2 intact, 2 ovx) and sham 2 operated (incision through skin only; 1 intact, 2 ovx) controls were also included in the study groups. Animals were evaluated on 3 consecutive days for withdrawal from touch with von Frey filaments on post-injury days 13-15, 20-22 and 27-29. A significant difference was observed at post-injury days 20-22 and 27-29 between the two groups. The intact group resulted in 12/12 animals that were statistically more sensitive than sham groups, compared to 6/12 for the ovx group. At post-injury days 13-15 no statistical difference was seen between the two groups (8/12 for intact versus 10/12 for ovx). This study indicates that ovarian hormones, via some unknown mechanism, predispose female rats to develop tactile allodynia following injury.

Orthodromic synaptic activation of rat olfactory bulb mitral cells in isolated slices.

Axons of olfactory receptor neurons terminate in the glomerular layer of the olfactory bulb, where they synapse with the apical dendrites of mitral cells. Although the mitral cell and its excitation by the olfactory nerve have been the subject of numerous experimental investigations, in vitro studies of these neurons have primarily used nonmammalian preparations. We have recorded the responses of rat olfactory bulb mitral cells to stimulation of the olfactory nerve layer in vitro using extracellular and whole cell patch techniques. Olfactory bulbs were cut into 400-microns thick slices in approximately horizontal section and submerged in a recording chamber. Patch clamp electrodes were guided into the mitral cell layer, which was visible under a dissecting microscope. A stimulating electrode was placed onto the olfactory nerve layer (ONL) rostral to the recording electrode. In extracellular recordings, mitral cells typically responded to ONL stimulation with a prolonged excitation lasting 1 s or longer. With whole cell patch recordings, membrane resistances (mean 272 M omega) were substantially higher than those reported in previous intracellular studies that used sharp electrodes. Small spontaneous excitatory potentials were present in some mitral cells. ONL stimulation caused a prolonged depolarization comparable to the duration of the period of excitation observed in extracellular recordings. At membrane potentials near -55 mV, ONL stimulation evoked a train of spikes. All but the first of these spikes were blocked by hyperpolarization of the membrane to -65 mV.

Medial preoptic area afferents to periaqueductal gray medullo-output neurons: a combined Fos and tract tracing study.

We have shown recently that the medial preoptic area (MPO) robustly innervates discrete columns along the rostrocaudal axis of the midbrain periaqueductal gray (PAG). However, the location of PAG neurons responsive to MPO activation is not known. Anterograde tract tracing was used in combination with Fos immunohistochemistry to characterize the MPO --> PAG pathway anatomically and functionally within the same animal. Focal electrical or chemical stimulation of MPO in anesthetized rats induced extensive Fos expression within the PAG compared with sham controls. Fos-positive neurons were organized as 2-3 longitudinal columns. The organization and location of these columns overlapped remarkably well with the distribution of fibers and terminals in PAG labeled by Phaseolus vulgaris leucoagglutinin (PHA-L) injected into the same MPO stimulation site. This indicates that MPO inputs may terminate on the soma or proximal dendrites of neurons exhibiting elevated Fos. A second series of experiments investigated whether MPO stimulation excited PAG neurons with descending projections to the medulla. Retrograde labelling of PAG neurons projecting to the medial and lateral regions of the rostroventral medulla (RVM) was combined with MPO-induced Fos expression. The results showed that a substantial population (37-53%) of Fos-positive PAG neurons projected to the ventral medulla. This indicates that MPO stimulation engages PAG-medullary output neurons. Taken together, these results suggest that the MPO --> Pag --> RVM projection constitutes a functional pathway. This circuit may coordinately regulate neuroendocrine, motor, and autonomic adjustments necessary for the elaboration of sexual behaviors.

Fos expression induced by changes in arterial pressure is localized in distinct, longitudinally organized columns of neurons in the rat midbrain periaqueductal gray.

The distribution of neurons expressing Fos within the periaqueductal gray (PAG) following pharmacologically induced high or low blood pressure was examined to determine (1) if PAG neurons are responsive to changes in arterial pressure (AP) and (2) the relationship of these cells to the functionally defined hypertensive and hypotensive columns in PAG. Changes in AP differentially induced robust Fos expression in neurons confined to discrete, longitudinally organized columns within PAG. Increased AP produced extensive Fos-like immunoreactivity within the lateral PAG, beginning at the level of the oculomotor nucleus. At the level of the dorsal raphe, Fos expression induced by increased AP shifted dorsally, into the dorsolateral division of PAG; this pattern of Fos labeling was maintained throughout the caudal one-third of PAG. Double-labeling for Fos and nicotinamide adenine dinucleotide phosphate diaphorase confirmed that Fos-positive cells induced by increased AP were located in the dorsolateral division of PAG at these caudal levels. Fos positive cells were codistributed, but not colocalized, with nicotinamide adenine dinucleotide phosphate diaphorase-positive cells. Decreased AP evoked a completely different pattern of Fos expression. Fos-positive cells were predominantly located within the ventrolateral PAG region, extending from the level of the trochlear nucleus through the level of the caudal dorsal raphe. Double-labeling studies for Fos and serotonin indicated that only 1-2 double-labeled cells per section were present. Saline infusion resulted in very few Fos-like immunoreactive cells, indicating that volume receptor activation does not account for Fos expression in PAG evoked by changes in AP. These results indicate that (1) substantial numbers of PAG neurons are excited by pharmacologically induced changes in AP and (2) excitatory barosensitive PAG neurons are anatomically segregated based on their responsiveness to a specific directional change in AP.

Functional characteristics of the midbrain periaqueductal gray.

The major functions of the midbrain periaqueductal gray (PAG), including pain and analgesia, fear and anxiety, vocalization, lordosis and cardiovascular control are considered in this review article. The PAG is an important site in ascending pain transmission. It receives afferents from nociceptive neurons in the spinal cord and sends projections to thalamic nuclei that process nociception. The PAG is also a major component of a descending pain inhibitory system. Activation of this system inhibits nociceptive neurons in the dorsal horn of the sinal cord. The dorsal PAG is a major site for processing of fear and anxiety. It interacts with the amygdala and its lesion alters fear and anxiety produced by stimulation of amygdala. Stimulation of PAG produces vocalization and its lesion produces mutism. The firing of many cells within the PAG correlates with vocalization. The PAG is a major site for lordosis and this role of PAG is mediated by a pathway connecting the medial preoptic with the PAG. The cardiovascular controlling network within the PAG are organized in columns. The dorsal column is involved in pressor and the ventrolateral column mediates depressor responses. The major intrinsic circuit within the PAG is a tonically-active GABAergic network and inhibition of this network is an important mechanism for activation of outputs of the PAG. The various functions of the PAG are interrelated and there is a significant interaction between different functional components of the PAG. Using the current information about the anatomy, physiology, and pharmacology of the PAG, a model is proposed to account for the interactions between these different functional components.

An electrophysiological characterization of the projection from the central nucleus of the amygdala to the periaqueductal gray of the rat: the role of opioid receptors.

The midbrain periaqueductal gray (PAG) and the central nucleus of the amygdala (CNA) are both known to be involved in fear and anxiety, analgesia, vocalization, cardiovascular and respiratory changes, and freezing. Anatomical studies have shown that a connection between these two regions exists but little is known about the physiology or the neurochemical constituents of this pathway. The goals of this study were to characterize the projection from the CNA to the PAG using electrophysiological techniques and to determine whether mu- and/or delta-opioid receptors, which play a large role in a majority of the functions of the PAG, are involved in this pathway. Of the 38 PAG cells tested with single shock stimulation of the CNA, 44% responded; of those, 46% were excited and 54% were inhibited. The latency to onset of response for the inhibitory cells (12.71 +/- 6.61 ms) was shorter than that of the excitatory cells (22.33 +/- 4.04 ms). Forty-six percent of the 129 PAG cells tested with train electrical stimulation of the CNA responded; 44% were excited and 56% were inhibited. Chemical stimulation of the CNA (10 mM D,L-homocysteic acid) produced similar results; 48% (62/128) of PAG cells responded; 45% of cells were excited and 55% were inhibited. The baseline firing rate of the inhibitory cells was significantly higher compared to the excitatory cells. Chemical stimulation of the CNA produced an increase in blood pressure in 12 animals, a decrease in two animals, and had no effect on the blood pressure of 68 animals. The blood pressure changes ranged between 8.5 and 26.3 mmHg with a mean of 16.2 +/- 2.2 mmHg. The effect of naloxone (given either on site in the PAG or systemically) on the response to CNA stimulation was tested in 21 cells. Twenty-five percent of the excitatory cells (2/8) and 77% (10/13) of the inhibitory cells were blocked by naloxone with the majority of the blocked cells located in the ventrolateral PAG. It is concluded that: (1) Approximately 50% of cells in the lateral and ventrolateral columns of the PAG respond to CNA stimulation; (2) the inhibitory response is mediated by a faster conducting or a more direct pathway than the pathway that mediates the excitatory response; (3) neurons that are inhibited by CNA stimulation have a significantly higher baseline firing rate than neurons that are excited, suggesting that they may be tonically active interneurons; and (4) at least one link in the CNA-PAG pathway utilizes mu- or delta-opioid receptors.

Directionally specific changes in arterial pressure induce differential patterns of fos expression in discrete areas of the rat brainstem: a double-labeling study for Fos and catecholamines.

Although the nucleus tractus solitarii (NTS) has been established as the primary site of synaptic integration for the baroreceptor reflex, the higher-order pathways responsive to, and mediating, changes in vasomotor tone are not well characterized. We used immunohistochemistry to determine the distribution of cells expressing the Fos protein following pharmacologically induced, directionally specific changes in arterial pressure. The goal of this investigation was to determine if this immediate early gene product is differentially expressed in neurons of the rat brainstem following increased (pressor) versus decreased (depressor) arterial blood pressure (AP). Because brainstem catecholaminergic (CA) cell groups have been implicated in cardiovascular regulation, a double-labeling immunohistochemical procedure was used to examine the distribution of Fos in CA cells. Animals received continuous intravenous infusion of either a vasoconstrictor (l-phenylephrine hydrochloride), a vasodilator (sodium nitroprusside), or physiological saline. Extensive Fos-like immunoreactivity (FLI) was induced in both the pressor and depressor conditions in the NTS, caudal ventrolateral medulla (CVLM), rostral ventrolateral medulla (RVLM), A5, locus coeruleus (LC), Kolliker-Fuse, and parabrachial nucleus (PBN). These regions have all been implicated in central cardiovascular regulation. There were differences in the anatomical distribution of Fos-positive cells along the rostrocaudal axis of CVLM in the pressor and depressor conditions. Specifically, increased AP induced significantly more FLI cells within the rostral aspects of CVLM, whereas decreased AP resulted in a significantly greater number of FLI cells within the caudal CVLM. This result suggests that selective vasomotor responses differentially engaged discrete subsets of neurons within this brainstem region. Overall, approximately 50% of CA-immunoreactive cells were also FLI (CA-FLI) in the A1, A5, and A7 regions. Interestingly, increased AP produced significantly more CA-FLI double-labeled cells within the caudal than rostral A1 compared with depressor and control groups. Additionally, increased AP yielded significantly less CA-FLI double-labeled cells within the caudal A2 region. This suggests that CA barosensitive neurons in the CVLM/A1 and NTS/A2 regions are functionally segregated along the rostrocaudal axis of these structures. While twice as many PNMT-FLI double-labeled neurons were found in the C1-C3 regions following vasomotor changes versus saline control, there were no differences in the numbers or anatomical locations of labeled cells between pressor versus depressor groups. The results of this study indicate that (1) tonic changes in AP induce robust Fos expression in brainstem cardiovascular areas and (2) neurons responsive to specific directional changes in arterial pressure are segregated in some brainstem regions.

The neurophysiologic mechanisms of tourniquet pain. The activity of neurons in the rostroventral medulla in the rat.

BACKGROUND: Tourniquet pain frequently complicates the use of pneumatic tourniquets during surgical procedures involving the extremities. The mechanisms of tourniquet pain are not understood and therefore the treatment is nonspecific. An animal model was sought to provide an ethical means to study the neurophysiologic and neuropharmacologic mechanisms of tourniquet pain. Neurons in the rostral ventromedial medulla are involved in the nociceptive-and circulatory-responsive neuronal networks. The goal of this study was to determine the activity of neurons in the rostral ventromedial medulla in response to the maintenance of tourniquet inflation in the rat as a component of an investigation of the neurophysiologic mechanisms of tourniquet-induced pain. METHODS: In 18 male, pentobarbital-anesthetized rats, heart rate, systolic blood pressure (SBP), and cell firing rates (CFR) of nociceptive-responsive rostral ventromedial medulla neurons, characterized as having an OFF or ON cell response to noxious heat, were monitored. CFR was monitored continuously at baseline, during an IV infusion of phenylephrine sufficient to increase SBP by 50%, during the application of a pneumatic tourniquet to the hind limb thigh and inflated to 300 mmHg for a period of 60 min, and during a 30-min recovery period. RESULTS: Phenylephrine-induced hypertension resulted in an increase in OFF CFR. Maintenance of tourniquet inflation resulted in a progressive decrease in OFF CFR and a progressive increase in ON CFR. An increase in SBP in response to tourniquet pain paralleled the changes in CFR. Mean SBP at 5 min preinflation, 5 min postinflation, 55 min postinflation, and 10 min postdeflation were 101 +/- 11, 103 +/- 9, 118 +/- 14, and 103 +/- 12 mmHg, respectively. CONCLUSIONS: The changes in SBP and CFR observed during tourniquet inflation were consistent with previously reported responses to nociceptive stimuli. Phenylephrine-induced hypertension caused an opposite effect on the CFR of rostral ventromedial medulla neurons as compared with a noxious stimulus such as heat or maintenance of tourniquet inflation. This experimental design is presented as an animal model to study the neurophysiologic and neuropharmacologic aspects of tourniquet pain.