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The effects of prostaglandin E2 on the firing rate activity of thermosensitive and temperature insensitive neurons in the ventromedial preoptic area of the rat hypothalamus
Brain Research 964 (2003) 42–50 www.elsevier.com / locate / brainres
Research report
The effects of prostaglandin E 2 on the firing rate activity of thermosensitive and temperature insensitive neurons in the ventromedial preoptic area of the rat hypothalamus Heather J. Ranels, John D. Griffin* Department of Biology, College of William and Mary, Williamsburg, VA 23187, USA Accepted 19 November 2002
Abstract In response to an immune system challenge with lipopolysaccharide (LPS), recent work has shown that Fos immunoreactivity is displayed by neurons in the ventromedial preoptic area of the hypothalamus (VMPO). In addition, neurons in this region show distinct axonal projections to the anterior perifornical area (APFx) and the paraventricular nucleus (PVN). It has been hypothesized that neurons within the VMPO integrate their local responses to temperature with changes in firing activity that result from LPS induced production of prostaglandin E 2 (PGE 2 ). This may be an important mechanism by which the set-point regulation of thermoeffector neurons in the APFx and PVN is altered, resulting in hyperthermia. To characterize the firing rate activity of VMPO neurons, single-unit recordings were made of neuronal extracellular activity in rat hypothalamic tissue slices. Based on the slope of firing rate as a function of tissue temperature, neurons were classified as either warm sensitive or temperature insensitive. Neurons were then treated with PGE 2 (200 nM) while tissue temperature was held at a constant level (|36 8C). The majority of temperature insensitive neurons responded to PGE 2 with an increase in firing rate activity, while warm sensitive neurons showed a reduction in firing rate. This suggests that both warm sensitive and temperature insensitive neurons in the VMPO may play critical and contrasting roles in the production of a fever during an acute phase response to infection. 2002 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Neural-immune interactions Keywords: Endogenous pyrogen; Fever; Hypothalamus; Prostaglandin E 2 ; Thermoregulation
1. Introduction The acute phase response is a multi-system coordinated reaction to immune stimulation that includes a wide variety of metabolic, endocrine, autonomic, and behavioral responses, which are controlled by the central nervous system [27]. Current theories suggest that these responses are triggered by the actions of circulating endogenous pyrogens on vagal sensory pathways, as well as by altering the activity of hypothalamic neurons that are in close proximity to the organum vasculosum of the lamina terminalis (OVLT) [2]. These varying pathways of activa*Corresponding author. Tel.: 11-757-221-2257; fax: 11-757-2216483. E-mail address: [email protected] (J.D. Griffin).
tion may allow for specific immune conditions to elicit unique patterns of stimulation. Fever, which is a component of the acute phase response, is an elevation in body temperature of 1–4 8C. A suggested mechanism for this response is a shift in the thermostatic set-point [4]. This set-point is achieved through the integration of a baseline level of non-thermal activation, with both central and afferent thermal information by neurons in the anterior regions of the hypothalamus. These neurons can be classified as either inherently thermosensitive or temperature insensitive and regulate the activity of efferent pathways to control thermoregulatory responses. During a fever, changes in the activity of neurons in the anterior hypothalamus may be the mechanism by which the thermostatic set-point is shifted into the hyperthermic range.
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )04063-5
H. J. Ranels, J.D. Griffin / Brain Research 964 (2003) 42–50
Several studies have shown that endotoxins such as lipopolysaccharide (LPS), a complex glycolipid found in the outer membrane of most gram-negative bacteria, stimulate leukocytes and other cells to produce certain endogenous substances that have pyrogenic effects [2]. These endogenous pyrogens may not act directly on hypothalamic neurons, but induce the production of prostaglandin E 2 (PGE 2 ) within specific regions of the hypothalamus. As early as the 1970s, microinjection studies established the role of prostaglandins in the production of fever [14,32]. Later, similar work clearly identified the region around the OVLT as being highly sensitive to PGE 2 and anatomically distinct in its ability to activate a febrile response to infection [1,33]. More recent studies have shown that in addition to a fever, intravenous injection of LPS resulted in an increased production within the hypothalamus of cyclooxygenase-2 (COX2), a primary enzyme in the synthesis of PGE 2 [29]. Further experiments using microinjections of keterolac (a COX2 inhibitor) identified discrete sites of activation that are necessary for fever. More specifically, this research identified a critical cell group adjacent to the OVLT that is known as the ventromedial preoptic area (VMPO). In addition to the physiologic evidence mentioned above, the importance of the VMPO as a site of fever induction has also been confirmed anatomically. In response to intravenous injection of LPS or microinjection of PGE 2 directly into the VMPO, neurons in this region showed a selective expression of Fos, an immediate early gene product that is present during increased cellular activation [12,28]. The ability of neurons in the VMPO to respond to the local production of PGE 2 is also supported by evidence that all four PGE 2 receptor subtypes are present within the hypothalamus [24]. Within the anterior regions of the hypothalamus, including the VMPO, there is overlapping expression of EP1 , EP3 and EP4 receptor subtypes [10]. With respect to the generation of a fever, recent work indicates that both EP3 and EP4 receptor subtypes are distinctly important [10,23,32]. If an increased concentration of PGE 2 in the anterior hypothalamus is responsible for the production of a fever, then a correlation should exist between the thermosensitivity of neurons in this region and responses to PGE 2 . Although several extracellular single-unit recording studies have characterized the effects of PGE 2 on the firing rate activity of hypothalamic neurons, no correlation with thermosensitivity has been reported. Matsuda et al. [20] showed that PGE 2 decreased the firing rates of some warm sensitive neurons, while having no effect or increasing the firing rates of other warm sensitive neurons. This conclusion is in contrast to similar studies, which indicate that some cytokines selectively decrease the firing rates of warm sensitive neurons [17,22]. However, these previous studies used varying criteria to define warm sensitivity and did not limit their recording locations to any specific functional nuclei within the anterior hypothalamus. Using
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a well established functional criteria to define thermosensitivity, we have characterized the firing rate activity of VMPO neurons in response to temperature and PGE 2 .
2. Materials and methods Hypothalamic tissue slices containing the VMPO were prepared from male Sprague–Dawley rats (100–150 g in weight), which were housed under standard conditions (23 8C; 12:12-h light:dark cycle, with lights on at 8.00 am) and given food and water ad lib. Prior to each recording session, an animal was anesthetized using isoflourene and sacrificed by decapitation, according to procedures approved by the Animal Care and Use Committee of the College of William and Mary. The brain was then quickly removed and a tissue block containing the hypothalamus was prepared. To ensure that recordings could be done from as much of the VMPO as possible, two or three 400 mm thick coronal slices of the anterior hypothalamus were sectioned and placed in a tissue chamber [18]. Slices were allowed to equilibrate for 2 h before recordings were attempted. Throughout the recording session, the tissue slices were perfused with a pyrogen free nutrient medium consisting of (mM): 124 NaCl, 26 NaHCO 3 , 10 glucose, 5 KCl, 2.4 CaCl 2 , 1.3 MgSO 4 and 1.24 KH 2 PO 4 . This medium was oxygenated (95% O 2 –5% CO 2 ), warmed to a constant temperature of approximately 36 8C by a thermoelectric assembly, and allowed to flow into the chamber at 1–1.5 ml min 21 [19]. A small thermocouple was positioned just below the tissue slices to continuously monitor temperature. Extracellular single-unit recordings were made from neurons in the VMPO using glass microelectrodes with tip diameters of less than 1 mm and filled with 3 M NaCl. All recordings were made using a Xcell-3 Microelectrode Amplifier (FHC Inc.) and stored along with temperature on tape for later analysis. Once the activity of a neuron was isolated (signal:noise $3:1) and stable for several minutes, changes in the input voltage to the thermoelectric assembly were used vary the temperature in the recording chamber 2–3 8C above and below 36 8C to determine responses to temperature. Neuronal thermosensitivity (impulses s 21 8C 21 ) was characterized by plotting firing rate as a function of temperature to determine the regression coefficient (m) of this plot. As in previous studies [15,18], warm sensitivity was defined as a regression coefficient of at least 0.8 impulses s 21 8C 21 . All other neurons in this study were defined as temperature insensitive. After the thermosensitivity of a neuron had been characterized, each neuron was tested for its response to PGE 2 . Once a stable temperature was achieved (|36 8C), the perfusion medium was switched to one containing PGE 2 (200 nM, Sigma Chemical Co.). The duration of exposure to PGE 2 ranged from 5 to 15 min. Treatment was stopped
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prior to 10 min only when a significant change in firing rate (impulses s 21 ) was recorded. Exposure to PGE 2 was followed by a control period of at least 10 min. After the recording session, one min samples of stable firing rate activity were digitized (60 Hz) for comparison (pClamp Software, Axon Instruments). These samples were collected during baseline conditions (just prior to perfusion with PGE 2 ), at the end of perfusion with PGE 2 or at the peak of a change in firing rate, and at the end of a 10 min control period or when firing rate returned to baseline levels. For each sample of firing rate activity, a mean and standard error were calculated (Sigmaplot Software, SPSS Inc.). Responses to PGE 2 were determined by comparison to baseline levels, using a standard T-test (P#0.05). A critical component of this study is that all recordings were made from neurons that were located in the VMPO. This region extends 700 mm rostrally from the suprachiasmatic nucleus, 900 mm laterally from the edge of the third ventricle, and 500 mm dorsally from the ventral surface of the brain [25]. In coronal tissue slices, this region can be visualized without difficulty. During each recording session, electrodes were placed within the VMPO, using stereoscopic visualization [7,9,30]. Once a recording had been completed, the location of the electrode was visually confirmed. The ventral edge of the third ventricle was used as a reference to determine lateral–medial and dorsal–ventral coordinates. In addition, the coronal position was further specified by recording the depth of the electrode from the surface of the tissue slice, and the side of the brain from which the recording was made was determined by preparing the tissue slices with more lateral tissue on the left side. Tissue slices were then removed from the recording chamber and fixed in a 10% formalin solution for at least 2 h. This was followed by at least 2 h in a 30% sucrose solution and then tissue slices were sectioned to a thickness of 50 mm. Once the tissue sections were mounted on gel coated microscope slides, a giemsa staining procedure was used to identify specific hypothalamic areas so that the location of each electrode within VMPO could be reconfirmed [15,25]. Only recordings from neurons within the VMPO were included in this study.
3. Results
3.1. Thermosensitivity The thermosensitivities of 51 VMPO neurons were characterized and their firing rate activities studied in response to PGE 2 . The majority of these neurons were classified as temperature insensitive (n541). All other neurons were classified as warm sensitive (n510). With respect to thermosensitivity, there was no specific pattern to the distribution of these neurons throughout the VMPO (Fig. 1).
3.2. Firing rate activity and responses to PGE2 The average baseline firing rate for all recorded VMPO neurons in this study was 4.6460.49 impulses s 21 . The average baseline firing rate of temperature insensitive neurons (4.0560.37 impulses s 21 ) was lower than the average baseline firing rate of warm sensitive neurons (7.0661.86 impulses s 21 ). However, this difference was not significant due to the large range in baseline firing rates recorded from warm sensitive neurons (1.87–17.18 impulses s 21 ). In response to PGE 2 , the majority (n525) of temperature insensitive neurons showed a significant increase in firing rate. As shown in Fig. 2, all of these neurons had thermosensitivities #0.4 impulses s 21 8C 21 . Three additional neurons, with similar thermosensitivities, showed little or no change in firing rate during perfusion with PGE 2 , while one neuron showed a significant decrease in firing rate. However, Table 1 indicates that the average firing rate activity of these 29 temperature insensitive neurons increased significantly in response to PGE 2 . Fig. 3 shows the firing rate activity of a temperature insensitive neuron (m50.01). In response to PGE 2 , firing rate increased 137%, from 4.9060.16 to 11.6360.31 impulses s 21 . The response took several minutes to develop and lasted approximately 20 min beyond the point when perfusion with PGE 2 was stopped. This was typical of all temperature insensitive neurons which responded to PGE 2 . The average latency until firing rate showed a significant increase was 5.2560.49 min. The duration of response to PGE 2 ranged from 5 to 44 min, with a few neurons (n57) not showing a return to a baseline level of firing rate during the control period. An additional twelve temperature insensitive neurons, which had thermosensitivies ranging from 0.41 to 0.79 impulses s 21 8C 21 , showed little or no change in firing rate in response to perfusion with PGE 2 (Fig. 2, Table 1). Fig. 4 shows the firing rate activity of a temperature insensitive neuron. After determining this neuron’s thermosensitivity (m50.54), PGE 2 was added to the perfusion medium for 10 min. Firing rate did not change significantly from the baseline level 8.5160.14 impulses s 21 . In response to PGE 2 , nine of the ten VMPO neurons classified as warm sensitive showed a significant decrease in firing rate (Fig. 2). Warm sensitive neurons decreased their firing rates an average of 57% in response to PGE 2 , with four neurons showing almost a complete inhibition of firing rate. As indicated in Table 1, the average firing rate activity of these of warm sensitive neurons decreased significantly in response to PGE 2 . Fig. 5 shows the firing rate activity of a warm sensitive neuron (m51.05). In response to PGE 2 , firing rate decreased 95.5%, from 1.5960.12 to 0.0760.03 impulses s 21 . The response took several minutes to develop and lasted throughout the control period. This was typical of all warm sensitive neurons which responded to PGE 2 . The
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Fig. 1. Extracellular single-unit recordings from VMPO Neurons. A shows a coronal diagram of the rat brain, at approximately the midpoint of the VMPO (modified from Fig. 19, Paxinos and Watson, 1998). In B, histological sections prepared from experimental tissue slices show the VMPO and surrounding hypothalamic areas. B II corresponds to the diagram in A, while B I and B III are rostral and caudal to B II , respectively. In C, coronal diagrams, which correspond to the histological sections in B, indicate the position of recorded neurons (insensitive neurons5•, warm sensitive neurons5m; modified diagrams from Paxinos and Watson, 1998). ac, anterior commisure; MnPo, median preoptic nucleus; BST, bed nucleus stria terminalis; 3V, third ventricle; Pe, periventricular hypothalamic nucleus; MPO, medial preoptic nucleus; LPO, lateral preoptic area; MPA, medial preoptic area; AVPe, anteroventral periventricular nucleus; mfb, medial forebrain bundle; VMPO, ventromedial preoptic nucleus; VLPO, ventrolateral preoptic nucleus; ox, optic chiasm.
Table 1 Effect of PGE 2 on the firing rate activity of VMPO neurons Thermosensitivity (impulses s 21 8C 21 )
N
#0.4 0.41–0.79 $0.8
29 12 10
Firing rate (impulses s 21 6S.E.) Baseline
PGE 2
Control
3.6460.41 5.0260.70 7.0661.86
6.3460.59* 4.9560.71 3.7961.75*
4.5360.55 4.9260.77 4.8961.77
*Significantly different from baseline firing rate (paired T-test, P,0.05).
average latency until firing rate showed a significant decrease was 7.1161.34 min. The duration of the response to PGE 2 ranged from 10 to 35 min, with 66% of warm sensitive neurons not showing a return to a baseline level of firing rate during the control period.
Fig. 2. The effects of PGE 2 on the firing rates of VMPO neurons. The percent change in firing rate (firing rate during perfusion with PGE 2 — baseline firing rate / baseline firing rate) is plotted for each neuron, with respect to thermosensitivity. Dotted line indicates a 0% change.
4. Discussion An enduring model of temperature regulation suggests that a set-point for body temperature is achieved through
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Fig. 3. The effects of temperature and PGE 2 on the firing rate activity of a VMPO temperature insensitive neuron. A shows the firing rate during changes in temperature and during perfusion with PGE 2 (indicated by the solid bar above the graph). In B, firing rate is plotted as a function of temperature. In C, one min samples of firing rate activity are plotted as individual bar graphs, just before perfusion with PGE 2 (Baseline; 4.9060.16) and during the peak of the response (PGE 2 ; 11.6360.31). For each plot in C, the error bars may be obscured.
the integration of a baseline level of non-thermal activation, with both central and afferent thermal information by neurons within the hypothalamus [16]. This model has been supported by numerous studies which indicate that the anterior hypothalamus has control over virtually all thermoregulatory responses and is sensitive to changes in local temperature [3]. Within this region, approximately 35% of the neurons can be classified as warm sensitive. These neurons are not only inherently thermosensitive, but many are influenced by peripheral temperature and show a direct correlation between their firing rate activity and the stimulation of specific thermoregulatory responses. Recent data also indicates that warm sensitive neurons receive local synaptic input primarily from temperature insensitive neurons [15]. This input may provide a non-thermal reference signal that contributes to the establishment of the thermostatic set-point. It can then be hypothesized that either an increase in the activity of temperature insensitive
neurons or a decrease in the activity of warm sensitive neurons could result in an adjustment of the set-point into the hyperthermic range. Although this model would suggest a strong correlation between inherent thermosensitivity and responses to PGE 2 , previous electrophysiology studies have not presented support for this theory. As stated earlier, this may have been due to the lack of a standard criteria for defining warm sensitivity and a general sampling of neurons from all regions of the anterior hypothalamus. Although previous investigations into the role of PGE 2 in fever have used criteria defining warm sensitivity which ranged from 0.5 to 0.7 impulses s 21 8C 21 [20–22], we have used a functional criteria of m$0.8 impulses s 21 8C 21 . This is based on in vivo recordings, which indicated that neurons meeting this functional criteria not only responded to local changes in temperature, but many were directly affected by afferent thermal input or showed a
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Fig. 4. The effect of temperature and PGE 2 on the firing rate activity of a VMPO temperature insensitive neuron. A shows the firing rate during changes in temperature and during perfusion with PGE 2 (indicated by the solid bar above the graph). In B, firing rate is plotted as a function of temperature. In C, one min samples of firing rate activity are plotted as individual bar graphs, just before perfusion with PGE 2 (Baseline; 8.5160.14) and during the peak of the response (PGE 2 ; 8.7160.13). For each plot in C, the error bars may be obscured.
correlation between firing rate and the activation of specific thermoregulatory responses [5,6]. Recent work also indicates that these warm sensitive neurons show a distinct pattern of dendritic morphology, which may be functionally significant [15]. It is also important to note that the majority of inherently warm sensitive neurons are located in the rostral hypothalamus, with large populations in the anterior and lateral hypothalamic areas, the medial preoptic area, and the APFx [8]. The majority of inherently cold sensitive neurons are found more caudally in the posterior hypothalamus, which may explain why no cold sensitive neurons were recorded in this study. This is the first study to specifically record the affects of temperature on the neuronal firing rate activity of VMPO neurons. Although we found that the VMPO had a smaller population of warm sensitive neurons compared to other regions of the rostral hypothalamus, all but one of these neurons
responded to PGE 2 with a significant decrease in firing rate (Fig. 2). Based on their responses to PGE 2 , the results of this study suggest that neurons functionally classified as temperature insensitive, form two distinct groups. All of the neurons with thermosensitivities of 0.41–0.79 impulses s 21 8C 21 did not respond significantly to PGE 2 , suggesting that they may not be directly involved in the stimulation of a fever through the activation of neurons in the VMPO (Fig. 2). The majority of temperature insensitive neurons with thermosensitivities #0.4 impulses s 21 8C 21 , responded to PGE 2 with a significant increase in firing rate (Fig. 2). As was seen with warm sensitive neurons, PGE 2 dependent changes in firing rate had a slow onset and lasted for at least 5 min, with most responses lasting considerably longer. In response to either electrophoretic application or perfusion of the PGE 2 dependent cytokine interleukin-1
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Fig. 5. The effect of temperature and PGE 2 on the firing rate activity of a VMPO warm sensitive neuron. A shows the firing rate during changes in temperature and during perfusion with PGE 2 (indicated by the solid bar above the graph). In B, firing rate is plotted as a function of temperature. In C, one min samples of firing rate activity are plotted as individual bar graphs, just before perfusion with PGE 2 (Baseline; 1.5960.12) and during the peak of the response (PGE 2 ; 0.0760.03). For each plot in C, the error bars may be obscured.
beta (IL-1), previous studies found that the responses of anterior hypothalamic neurons showed similar latencies and durations [17,31]. This might suggest that PGE 2 stimulates an indirect mechanism of cellular activation, such as is typical with the activation of a second messenger pathway. The activation of EP3 and EP4 receptor subtypes, which have been linked to the development of a fever and are present in the VMPO, have been shown to decrease intracellular cAMP or increase intracellular cAMP, respectively [10,23,24]. The elevation of body temperature which is typical of a fever is thought to be mediated by endogenous cytokines that are produced in response to infection. Some of these cytokines may have direct effects on the activity of neurons in the anterior hypothalamus. However, their ability to generate a fever is dependent on the local production of PGE 2 . In addition, the hyperthermic re-
sponse to pyrogenic activation of hepatic vagal afferents, which stimulate the release of norepinephrine in the anterior hypothalamus, has also been shown to be PGE 2 dependent [2]. Although neurons throughout the anterior hypothalamus may be responsive to endogenous substances such as PGE 2 , there is evidence that discrete neuronal populations within this region may specifically regulate homeostatic mechanisms, such as those involved in temperature regulation. With respect to the acute phase response to infection, neural activation of the VMPO has been established as a critical step in the production of a fever [13]. In addition, two distinct groups of LPS induced Fos activated neurons in the VMPO have been characterized, which form local efferent projections that may underlie the adjustment of the thermostatic set-point that results in fever. The larger of these two efferent pathways from the
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VMPO is an inhibitory projection to the anterior perifornical region (APFx), an area of the hypothalamus which contains a high proportion of warm sensitive neurons [8,11]. From the APFx, Roland and Sawchencko [26] have shown that there is a similar inhibitory projection to the autonomic parvicellular division of the PVN. Therefore, activation of this efferent pathway from the VMPO may result in the inhibition of warm sensitive neurons in the APFx, thereby decreasing the level of inhibition to the PVN. Consistent with this hypothesis and the current model of set-point thermoregulation, our data would suggest that this efferent pathway consists of temperature insensitive neurons from the VMPO that respond to PGE 2 with an increase in firing rate activity (Table 1). A more restricted efferent projection also exists directly from the VMPO to the PVN [11]. In contrast to previous electrophysiology studies of the anterior hypothalamus, we found that only a small percentage of neurons in the VMPO are warm sensitive. We also report that all of these warm sensitive neurons responded to PGE 2 with a significant decrease in firing rate (Table 1). Thus, it may be suggested that these warm sensitive neurons provide excitatory input to the PVN through this direct efferent projection. In response to increased concentrations of PGE 2 in the VMPO, the level of excitation would be reduced which may limit the activation of heat loss thermoregulatory responses (i.e., vasodilation) and result in a fever.
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Acknowledgements
[19]
We want to thank Erin Waller and Sarah Norcross for their assistance in the histological processing of neural tissue. This work was supported by NSF grant IBN9983624.
[20]
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Research report
The effects of prostaglandin E 2 on the firing rate activity of thermosensitive and temperature insensitive neurons in the ventromedial preoptic area of the rat hypothalamus Heather J. Ranels, John D. Griffin* Department of Biology, College of William and Mary, Williamsburg, VA 23187, USA Accepted 19 November 2002
Abstract In response to an immune system challenge with lipopolysaccharide (LPS), recent work has shown that Fos immunoreactivity is displayed by neurons in the ventromedial preoptic area of the hypothalamus (VMPO). In addition, neurons in this region show distinct axonal projections to the anterior perifornical area (APFx) and the paraventricular nucleus (PVN). It has been hypothesized that neurons within the VMPO integrate their local responses to temperature with changes in firing activity that result from LPS induced production of prostaglandin E 2 (PGE 2 ). This may be an important mechanism by which the set-point regulation of thermoeffector neurons in the APFx and PVN is altered, resulting in hyperthermia. To characterize the firing rate activity of VMPO neurons, single-unit recordings were made of neuronal extracellular activity in rat hypothalamic tissue slices. Based on the slope of firing rate as a function of tissue temperature, neurons were classified as either warm sensitive or temperature insensitive. Neurons were then treated with PGE 2 (200 nM) while tissue temperature was held at a constant level (|36 8C). The majority of temperature insensitive neurons responded to PGE 2 with an increase in firing rate activity, while warm sensitive neurons showed a reduction in firing rate. This suggests that both warm sensitive and temperature insensitive neurons in the VMPO may play critical and contrasting roles in the production of a fever during an acute phase response to infection. 2002 Elsevier Science B.V. All rights reserved. Theme: Endocrine and autonomic regulation Topic: Neural-immune interactions Keywords: Endogenous pyrogen; Fever; Hypothalamus; Prostaglandin E 2 ; Thermoregulation
1. Introduction The acute phase response is a multi-system coordinated reaction to immune stimulation that includes a wide variety of metabolic, endocrine, autonomic, and behavioral responses, which are controlled by the central nervous system [27]. Current theories suggest that these responses are triggered by the actions of circulating endogenous pyrogens on vagal sensory pathways, as well as by altering the activity of hypothalamic neurons that are in close proximity to the organum vasculosum of the lamina terminalis (OVLT) [2]. These varying pathways of activa*Corresponding author. Tel.: 11-757-221-2257; fax: 11-757-2216483. E-mail address: [email protected] (J.D. Griffin).
tion may allow for specific immune conditions to elicit unique patterns of stimulation. Fever, which is a component of the acute phase response, is an elevation in body temperature of 1–4 8C. A suggested mechanism for this response is a shift in the thermostatic set-point [4]. This set-point is achieved through the integration of a baseline level of non-thermal activation, with both central and afferent thermal information by neurons in the anterior regions of the hypothalamus. These neurons can be classified as either inherently thermosensitive or temperature insensitive and regulate the activity of efferent pathways to control thermoregulatory responses. During a fever, changes in the activity of neurons in the anterior hypothalamus may be the mechanism by which the thermostatic set-point is shifted into the hyperthermic range.
0006-8993 / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )04063-5
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Several studies have shown that endotoxins such as lipopolysaccharide (LPS), a complex glycolipid found in the outer membrane of most gram-negative bacteria, stimulate leukocytes and other cells to produce certain endogenous substances that have pyrogenic effects [2]. These endogenous pyrogens may not act directly on hypothalamic neurons, but induce the production of prostaglandin E 2 (PGE 2 ) within specific regions of the hypothalamus. As early as the 1970s, microinjection studies established the role of prostaglandins in the production of fever [14,32]. Later, similar work clearly identified the region around the OVLT as being highly sensitive to PGE 2 and anatomically distinct in its ability to activate a febrile response to infection [1,33]. More recent studies have shown that in addition to a fever, intravenous injection of LPS resulted in an increased production within the hypothalamus of cyclooxygenase-2 (COX2), a primary enzyme in the synthesis of PGE 2 [29]. Further experiments using microinjections of keterolac (a COX2 inhibitor) identified discrete sites of activation that are necessary for fever. More specifically, this research identified a critical cell group adjacent to the OVLT that is known as the ventromedial preoptic area (VMPO). In addition to the physiologic evidence mentioned above, the importance of the VMPO as a site of fever induction has also been confirmed anatomically. In response to intravenous injection of LPS or microinjection of PGE 2 directly into the VMPO, neurons in this region showed a selective expression of Fos, an immediate early gene product that is present during increased cellular activation [12,28]. The ability of neurons in the VMPO to respond to the local production of PGE 2 is also supported by evidence that all four PGE 2 receptor subtypes are present within the hypothalamus [24]. Within the anterior regions of the hypothalamus, including the VMPO, there is overlapping expression of EP1 , EP3 and EP4 receptor subtypes [10]. With respect to the generation of a fever, recent work indicates that both EP3 and EP4 receptor subtypes are distinctly important [10,23,32]. If an increased concentration of PGE 2 in the anterior hypothalamus is responsible for the production of a fever, then a correlation should exist between the thermosensitivity of neurons in this region and responses to PGE 2 . Although several extracellular single-unit recording studies have characterized the effects of PGE 2 on the firing rate activity of hypothalamic neurons, no correlation with thermosensitivity has been reported. Matsuda et al. [20] showed that PGE 2 decreased the firing rates of some warm sensitive neurons, while having no effect or increasing the firing rates of other warm sensitive neurons. This conclusion is in contrast to similar studies, which indicate that some cytokines selectively decrease the firing rates of warm sensitive neurons [17,22]. However, these previous studies used varying criteria to define warm sensitivity and did not limit their recording locations to any specific functional nuclei within the anterior hypothalamus. Using
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a well established functional criteria to define thermosensitivity, we have characterized the firing rate activity of VMPO neurons in response to temperature and PGE 2 .
2. Materials and methods Hypothalamic tissue slices containing the VMPO were prepared from male Sprague–Dawley rats (100–150 g in weight), which were housed under standard conditions (23 8C; 12:12-h light:dark cycle, with lights on at 8.00 am) and given food and water ad lib. Prior to each recording session, an animal was anesthetized using isoflourene and sacrificed by decapitation, according to procedures approved by the Animal Care and Use Committee of the College of William and Mary. The brain was then quickly removed and a tissue block containing the hypothalamus was prepared. To ensure that recordings could be done from as much of the VMPO as possible, two or three 400 mm thick coronal slices of the anterior hypothalamus were sectioned and placed in a tissue chamber [18]. Slices were allowed to equilibrate for 2 h before recordings were attempted. Throughout the recording session, the tissue slices were perfused with a pyrogen free nutrient medium consisting of (mM): 124 NaCl, 26 NaHCO 3 , 10 glucose, 5 KCl, 2.4 CaCl 2 , 1.3 MgSO 4 and 1.24 KH 2 PO 4 . This medium was oxygenated (95% O 2 –5% CO 2 ), warmed to a constant temperature of approximately 36 8C by a thermoelectric assembly, and allowed to flow into the chamber at 1–1.5 ml min 21 [19]. A small thermocouple was positioned just below the tissue slices to continuously monitor temperature. Extracellular single-unit recordings were made from neurons in the VMPO using glass microelectrodes with tip diameters of less than 1 mm and filled with 3 M NaCl. All recordings were made using a Xcell-3 Microelectrode Amplifier (FHC Inc.) and stored along with temperature on tape for later analysis. Once the activity of a neuron was isolated (signal:noise $3:1) and stable for several minutes, changes in the input voltage to the thermoelectric assembly were used vary the temperature in the recording chamber 2–3 8C above and below 36 8C to determine responses to temperature. Neuronal thermosensitivity (impulses s 21 8C 21 ) was characterized by plotting firing rate as a function of temperature to determine the regression coefficient (m) of this plot. As in previous studies [15,18], warm sensitivity was defined as a regression coefficient of at least 0.8 impulses s 21 8C 21 . All other neurons in this study were defined as temperature insensitive. After the thermosensitivity of a neuron had been characterized, each neuron was tested for its response to PGE 2 . Once a stable temperature was achieved (|36 8C), the perfusion medium was switched to one containing PGE 2 (200 nM, Sigma Chemical Co.). The duration of exposure to PGE 2 ranged from 5 to 15 min. Treatment was stopped
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prior to 10 min only when a significant change in firing rate (impulses s 21 ) was recorded. Exposure to PGE 2 was followed by a control period of at least 10 min. After the recording session, one min samples of stable firing rate activity were digitized (60 Hz) for comparison (pClamp Software, Axon Instruments). These samples were collected during baseline conditions (just prior to perfusion with PGE 2 ), at the end of perfusion with PGE 2 or at the peak of a change in firing rate, and at the end of a 10 min control period or when firing rate returned to baseline levels. For each sample of firing rate activity, a mean and standard error were calculated (Sigmaplot Software, SPSS Inc.). Responses to PGE 2 were determined by comparison to baseline levels, using a standard T-test (P#0.05). A critical component of this study is that all recordings were made from neurons that were located in the VMPO. This region extends 700 mm rostrally from the suprachiasmatic nucleus, 900 mm laterally from the edge of the third ventricle, and 500 mm dorsally from the ventral surface of the brain [25]. In coronal tissue slices, this region can be visualized without difficulty. During each recording session, electrodes were placed within the VMPO, using stereoscopic visualization [7,9,30]. Once a recording had been completed, the location of the electrode was visually confirmed. The ventral edge of the third ventricle was used as a reference to determine lateral–medial and dorsal–ventral coordinates. In addition, the coronal position was further specified by recording the depth of the electrode from the surface of the tissue slice, and the side of the brain from which the recording was made was determined by preparing the tissue slices with more lateral tissue on the left side. Tissue slices were then removed from the recording chamber and fixed in a 10% formalin solution for at least 2 h. This was followed by at least 2 h in a 30% sucrose solution and then tissue slices were sectioned to a thickness of 50 mm. Once the tissue sections were mounted on gel coated microscope slides, a giemsa staining procedure was used to identify specific hypothalamic areas so that the location of each electrode within VMPO could be reconfirmed [15,25]. Only recordings from neurons within the VMPO were included in this study.
3. Results
3.1. Thermosensitivity The thermosensitivities of 51 VMPO neurons were characterized and their firing rate activities studied in response to PGE 2 . The majority of these neurons were classified as temperature insensitive (n541). All other neurons were classified as warm sensitive (n510). With respect to thermosensitivity, there was no specific pattern to the distribution of these neurons throughout the VMPO (Fig. 1).
3.2. Firing rate activity and responses to PGE2 The average baseline firing rate for all recorded VMPO neurons in this study was 4.6460.49 impulses s 21 . The average baseline firing rate of temperature insensitive neurons (4.0560.37 impulses s 21 ) was lower than the average baseline firing rate of warm sensitive neurons (7.0661.86 impulses s 21 ). However, this difference was not significant due to the large range in baseline firing rates recorded from warm sensitive neurons (1.87–17.18 impulses s 21 ). In response to PGE 2 , the majority (n525) of temperature insensitive neurons showed a significant increase in firing rate. As shown in Fig. 2, all of these neurons had thermosensitivities #0.4 impulses s 21 8C 21 . Three additional neurons, with similar thermosensitivities, showed little or no change in firing rate during perfusion with PGE 2 , while one neuron showed a significant decrease in firing rate. However, Table 1 indicates that the average firing rate activity of these 29 temperature insensitive neurons increased significantly in response to PGE 2 . Fig. 3 shows the firing rate activity of a temperature insensitive neuron (m50.01). In response to PGE 2 , firing rate increased 137%, from 4.9060.16 to 11.6360.31 impulses s 21 . The response took several minutes to develop and lasted approximately 20 min beyond the point when perfusion with PGE 2 was stopped. This was typical of all temperature insensitive neurons which responded to PGE 2 . The average latency until firing rate showed a significant increase was 5.2560.49 min. The duration of response to PGE 2 ranged from 5 to 44 min, with a few neurons (n57) not showing a return to a baseline level of firing rate during the control period. An additional twelve temperature insensitive neurons, which had thermosensitivies ranging from 0.41 to 0.79 impulses s 21 8C 21 , showed little or no change in firing rate in response to perfusion with PGE 2 (Fig. 2, Table 1). Fig. 4 shows the firing rate activity of a temperature insensitive neuron. After determining this neuron’s thermosensitivity (m50.54), PGE 2 was added to the perfusion medium for 10 min. Firing rate did not change significantly from the baseline level 8.5160.14 impulses s 21 . In response to PGE 2 , nine of the ten VMPO neurons classified as warm sensitive showed a significant decrease in firing rate (Fig. 2). Warm sensitive neurons decreased their firing rates an average of 57% in response to PGE 2 , with four neurons showing almost a complete inhibition of firing rate. As indicated in Table 1, the average firing rate activity of these of warm sensitive neurons decreased significantly in response to PGE 2 . Fig. 5 shows the firing rate activity of a warm sensitive neuron (m51.05). In response to PGE 2 , firing rate decreased 95.5%, from 1.5960.12 to 0.0760.03 impulses s 21 . The response took several minutes to develop and lasted throughout the control period. This was typical of all warm sensitive neurons which responded to PGE 2 . The
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Fig. 1. Extracellular single-unit recordings from VMPO Neurons. A shows a coronal diagram of the rat brain, at approximately the midpoint of the VMPO (modified from Fig. 19, Paxinos and Watson, 1998). In B, histological sections prepared from experimental tissue slices show the VMPO and surrounding hypothalamic areas. B II corresponds to the diagram in A, while B I and B III are rostral and caudal to B II , respectively. In C, coronal diagrams, which correspond to the histological sections in B, indicate the position of recorded neurons (insensitive neurons5•, warm sensitive neurons5m; modified diagrams from Paxinos and Watson, 1998). ac, anterior commisure; MnPo, median preoptic nucleus; BST, bed nucleus stria terminalis; 3V, third ventricle; Pe, periventricular hypothalamic nucleus; MPO, medial preoptic nucleus; LPO, lateral preoptic area; MPA, medial preoptic area; AVPe, anteroventral periventricular nucleus; mfb, medial forebrain bundle; VMPO, ventromedial preoptic nucleus; VLPO, ventrolateral preoptic nucleus; ox, optic chiasm.
Table 1 Effect of PGE 2 on the firing rate activity of VMPO neurons Thermosensitivity (impulses s 21 8C 21 )
N
#0.4 0.41–0.79 $0.8
29 12 10
Firing rate (impulses s 21 6S.E.) Baseline
PGE 2
Control
3.6460.41 5.0260.70 7.0661.86
6.3460.59* 4.9560.71 3.7961.75*
4.5360.55 4.9260.77 4.8961.77
*Significantly different from baseline firing rate (paired T-test, P,0.05).
average latency until firing rate showed a significant decrease was 7.1161.34 min. The duration of the response to PGE 2 ranged from 10 to 35 min, with 66% of warm sensitive neurons not showing a return to a baseline level of firing rate during the control period.
Fig. 2. The effects of PGE 2 on the firing rates of VMPO neurons. The percent change in firing rate (firing rate during perfusion with PGE 2 — baseline firing rate / baseline firing rate) is plotted for each neuron, with respect to thermosensitivity. Dotted line indicates a 0% change.
4. Discussion An enduring model of temperature regulation suggests that a set-point for body temperature is achieved through
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Fig. 3. The effects of temperature and PGE 2 on the firing rate activity of a VMPO temperature insensitive neuron. A shows the firing rate during changes in temperature and during perfusion with PGE 2 (indicated by the solid bar above the graph). In B, firing rate is plotted as a function of temperature. In C, one min samples of firing rate activity are plotted as individual bar graphs, just before perfusion with PGE 2 (Baseline; 4.9060.16) and during the peak of the response (PGE 2 ; 11.6360.31). For each plot in C, the error bars may be obscured.
the integration of a baseline level of non-thermal activation, with both central and afferent thermal information by neurons within the hypothalamus [16]. This model has been supported by numerous studies which indicate that the anterior hypothalamus has control over virtually all thermoregulatory responses and is sensitive to changes in local temperature [3]. Within this region, approximately 35% of the neurons can be classified as warm sensitive. These neurons are not only inherently thermosensitive, but many are influenced by peripheral temperature and show a direct correlation between their firing rate activity and the stimulation of specific thermoregulatory responses. Recent data also indicates that warm sensitive neurons receive local synaptic input primarily from temperature insensitive neurons [15]. This input may provide a non-thermal reference signal that contributes to the establishment of the thermostatic set-point. It can then be hypothesized that either an increase in the activity of temperature insensitive
neurons or a decrease in the activity of warm sensitive neurons could result in an adjustment of the set-point into the hyperthermic range. Although this model would suggest a strong correlation between inherent thermosensitivity and responses to PGE 2 , previous electrophysiology studies have not presented support for this theory. As stated earlier, this may have been due to the lack of a standard criteria for defining warm sensitivity and a general sampling of neurons from all regions of the anterior hypothalamus. Although previous investigations into the role of PGE 2 in fever have used criteria defining warm sensitivity which ranged from 0.5 to 0.7 impulses s 21 8C 21 [20–22], we have used a functional criteria of m$0.8 impulses s 21 8C 21 . This is based on in vivo recordings, which indicated that neurons meeting this functional criteria not only responded to local changes in temperature, but many were directly affected by afferent thermal input or showed a
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Fig. 4. The effect of temperature and PGE 2 on the firing rate activity of a VMPO temperature insensitive neuron. A shows the firing rate during changes in temperature and during perfusion with PGE 2 (indicated by the solid bar above the graph). In B, firing rate is plotted as a function of temperature. In C, one min samples of firing rate activity are plotted as individual bar graphs, just before perfusion with PGE 2 (Baseline; 8.5160.14) and during the peak of the response (PGE 2 ; 8.7160.13). For each plot in C, the error bars may be obscured.
correlation between firing rate and the activation of specific thermoregulatory responses [5,6]. Recent work also indicates that these warm sensitive neurons show a distinct pattern of dendritic morphology, which may be functionally significant [15]. It is also important to note that the majority of inherently warm sensitive neurons are located in the rostral hypothalamus, with large populations in the anterior and lateral hypothalamic areas, the medial preoptic area, and the APFx [8]. The majority of inherently cold sensitive neurons are found more caudally in the posterior hypothalamus, which may explain why no cold sensitive neurons were recorded in this study. This is the first study to specifically record the affects of temperature on the neuronal firing rate activity of VMPO neurons. Although we found that the VMPO had a smaller population of warm sensitive neurons compared to other regions of the rostral hypothalamus, all but one of these neurons
responded to PGE 2 with a significant decrease in firing rate (Fig. 2). Based on their responses to PGE 2 , the results of this study suggest that neurons functionally classified as temperature insensitive, form two distinct groups. All of the neurons with thermosensitivities of 0.41–0.79 impulses s 21 8C 21 did not respond significantly to PGE 2 , suggesting that they may not be directly involved in the stimulation of a fever through the activation of neurons in the VMPO (Fig. 2). The majority of temperature insensitive neurons with thermosensitivities #0.4 impulses s 21 8C 21 , responded to PGE 2 with a significant increase in firing rate (Fig. 2). As was seen with warm sensitive neurons, PGE 2 dependent changes in firing rate had a slow onset and lasted for at least 5 min, with most responses lasting considerably longer. In response to either electrophoretic application or perfusion of the PGE 2 dependent cytokine interleukin-1
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Fig. 5. The effect of temperature and PGE 2 on the firing rate activity of a VMPO warm sensitive neuron. A shows the firing rate during changes in temperature and during perfusion with PGE 2 (indicated by the solid bar above the graph). In B, firing rate is plotted as a function of temperature. In C, one min samples of firing rate activity are plotted as individual bar graphs, just before perfusion with PGE 2 (Baseline; 1.5960.12) and during the peak of the response (PGE 2 ; 0.0760.03). For each plot in C, the error bars may be obscured.
beta (IL-1), previous studies found that the responses of anterior hypothalamic neurons showed similar latencies and durations [17,31]. This might suggest that PGE 2 stimulates an indirect mechanism of cellular activation, such as is typical with the activation of a second messenger pathway. The activation of EP3 and EP4 receptor subtypes, which have been linked to the development of a fever and are present in the VMPO, have been shown to decrease intracellular cAMP or increase intracellular cAMP, respectively [10,23,24]. The elevation of body temperature which is typical of a fever is thought to be mediated by endogenous cytokines that are produced in response to infection. Some of these cytokines may have direct effects on the activity of neurons in the anterior hypothalamus. However, their ability to generate a fever is dependent on the local production of PGE 2 . In addition, the hyperthermic re-
sponse to pyrogenic activation of hepatic vagal afferents, which stimulate the release of norepinephrine in the anterior hypothalamus, has also been shown to be PGE 2 dependent [2]. Although neurons throughout the anterior hypothalamus may be responsive to endogenous substances such as PGE 2 , there is evidence that discrete neuronal populations within this region may specifically regulate homeostatic mechanisms, such as those involved in temperature regulation. With respect to the acute phase response to infection, neural activation of the VMPO has been established as a critical step in the production of a fever [13]. In addition, two distinct groups of LPS induced Fos activated neurons in the VMPO have been characterized, which form local efferent projections that may underlie the adjustment of the thermostatic set-point that results in fever. The larger of these two efferent pathways from the
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VMPO is an inhibitory projection to the anterior perifornical region (APFx), an area of the hypothalamus which contains a high proportion of warm sensitive neurons [8,11]. From the APFx, Roland and Sawchencko [26] have shown that there is a similar inhibitory projection to the autonomic parvicellular division of the PVN. Therefore, activation of this efferent pathway from the VMPO may result in the inhibition of warm sensitive neurons in the APFx, thereby decreasing the level of inhibition to the PVN. Consistent with this hypothesis and the current model of set-point thermoregulation, our data would suggest that this efferent pathway consists of temperature insensitive neurons from the VMPO that respond to PGE 2 with an increase in firing rate activity (Table 1). A more restricted efferent projection also exists directly from the VMPO to the PVN [11]. In contrast to previous electrophysiology studies of the anterior hypothalamus, we found that only a small percentage of neurons in the VMPO are warm sensitive. We also report that all of these warm sensitive neurons responded to PGE 2 with a significant decrease in firing rate (Table 1). Thus, it may be suggested that these warm sensitive neurons provide excitatory input to the PVN through this direct efferent projection. In response to increased concentrations of PGE 2 in the VMPO, the level of excitation would be reduced which may limit the activation of heat loss thermoregulatory responses (i.e., vasodilation) and result in a fever.
[8] [9]
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[17]
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Acknowledgements
[19]
We want to thank Erin Waller and Sarah Norcross for their assistance in the histological processing of neural tissue. This work was supported by NSF grant IBN9983624.
[20]
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