NaV1.7: Stress-induced changes in immunoreactivity within magnocellular neurosecretory neurons of the supraoptic nucleus

Background NaV1.7 is preferentially expressed, at relatively high levels, in peripheral neurons, and is often referred to as a “peripheral” sodium channel, and NaV1.7-specific blockers are under study as potential pain therapeutics which might be expected to have minimal CNS side effects. However, occasional reports of patients with NaV1.7 gain-of-function mutations and apparent hypothalamic dysfunction have appeared. The two sodium channels previously studied within the rat hypothalamic supraoptic nucleus, NaV1.2 and NaV1.6, display up-regulated expression in response to osmotic stress. Results Here we show that NaV1.7 is present within vasopressin-producing neurons and oxytocin-producing neurons within the rat hypothalamus, and demonstrate that the level of Nav1.7 immunoreactivity is increased in these cells in response to osmotic stress. Conclusions NaV1.7 is present within neurosecretory neurons of rat supraoptic nucleus, where the level of immunoreactivity is dynamic, increasing in response to osmotic stress. Whether NaV1.7 levels are up-regulated within the human hypothalamus in response to environmental factors or stress, and whether NaV1.7 plays a functional role in human hypothalamus, is not yet known. Until these questions are resolved, the present findings suggest the need for careful assessment of hypothalamic function in patients with NaV1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as NaV1.7 blocking agents are studied.


Background
Gain-of-function mutations of the Na V 1.7 sodium channel, which is preferentially expressed at relatively high levels within peripheral (dorsal root ganglion and sympathetic ganglion) neurons [1][2][3] produce several syndromes associated with severe pain, including inherited erythromelalgia [4][5][6][7][8] and paroxysmal extreme pain disorder [9,10] as well as painful small-fiber neuropathy [11,12], while loss-of-function mutations of Na V 1.7 cause channelopathy-associated insensitivity to pain [13][14][15]. In contrast with the severe pain associated with gain-of-function mutations of Na V 1.7 and loss of pain sensitivity associated with loss-of-function mutations of Na V 1.7, abnormalities of CNS function have in general not been reported in these disorders, consistent with preferential expression of Na V 1.7 within peripheral neurons. Na V 1.7specific blockers are being studied as potential therapies for pain, with the rationale that they would be expected to have few, if any, CNS-related side-effects. Nevertheless, there have been reports of hypothermia, possibly due to an abnormality of central (hypothalamic) thermoregulation [16][17][18] in patients with Na V 1.7 mutations and erythromelalgia. The syndrome of inappropriate release of antidiuretic hormone, SIADH, without any structural cause, recently developed in a patient carrying a gain-of-function mutation of Na V 1.7, G856D, within a kindred with painful small-fiber neuropathy (Hoeijmakers et al, personal communication). Affected family members, all of whom carry the G856D mutation, display small-fiber neuropathy characterized by severe pain and vasomotor dyscontrol in their distal extremities, small hands and feet, and autonomic dysfunction. The G856D mutation enhances channel activation, impairs fast-inactivation, and markedly enhances the channel's persistent current and response to slow ramp stimuli. The occurrence of SIADH in this patient suggested the possibility that the gain-of -function mutation in Na V 1.7 might have contributed to hyperexcitability of vasopressin-releasing (magnocellular neurosecretory) neurons in the supraoptic nucleus within the hypothalamus.
Vasopressin release by supraoptic magnocellular neurons can be triggered by osmotic stress and depends on bursting activity in these cells [19]. It is known that tetrodotoxin-sensitive sodium channels contribute to this bursting [20][21][22]. While high levels of expression of Na V 1.7 have been reported in hypothalamic nuclei including the supraoptic nucleus in rodents [13,23], only weak levels of Na V 1.7 expression were detected within the primate supraoptic nucleus [13]. In the present study, we have built upon earlier studies in rodents which showed that the deployment of sodium channels in the hypothalamus is dynamic, with levels of expression of the two sodium channel subtypes that were previously studied, Na V 1.2 and Na V 1.6, and of sodium channel beta-1 and beta-2 subunits and sodium currents, displaying upregulation within supraoptic magnocellular neurons exposed to osmotic stress via salt-loading [24] and as a result of the hyperosmolar state associated with experimental diabetes [25]. Reasoning that Na v 1.7 expression within supraoptic magnocellular neurons might be subject to similar plasticity, we exposed rats to salt-loading and assessed the level of Na V 1.7 immunoreactivity within these neurons. We demonstrate here that Na V 1.7 is present within vasopressin-and oxytocin-producing neurons of the supraoptic nucleus, and show that the level of Na V 1.7 protein in these cells in not static but, on the contrary, is increased in response to salt-loading.

Results
Previous work from our laboratory has demonstrated the expression of the tetrodotoxin-sensitive (TTX-S) sodium channels, Na V 1.2 and Na V 1.6, but not Na V 1.1 and Na V 1.3, and of TTX-S sodium currents in magnocellular neurosecretory cells (MSC) of the hypothalamic supraoptic nucleus [24]. This early study also showed that the expression of Na V 1.2 and Na V 1.6 channels are upregulated and amplitude of the sodium current increased following salt-loading challenge [24]. To determine whether Na V 1.7 is expressed and upregulated in magnocellular neurosecretory cells of the supraoptic nucleus, we assessed the supraoptic nucleus of control and salt-loaded (2% NaCl in drinking water) rats using immunocytochemistry. Measurement of plasma osmotic pressure confirmed the presence of hyperosmolarity in the salt-loaded rats: control, 323.3 ± 4.8 mOsm; salt-loaded, 353.2 ± 3.3 mOsm (p < 0.05).
Magnocellular neurosecretory cells in the supraoptic nucleus of control rats exhibited distinct Na V 1.7 immunolabeling (Figure 1). Some magnocellular neurosecretory cells displayed moderate levels of Na V 1.7 immunosignal, while other magnocellular neurosecretory cells exhibited a low level or no Na V 1.7 immunofluorescence. Two types of magnocellular neurosecretory cells exist within the supraoptic nucleus, oxytocin-producing and vasopressin-producing, with little co-expression of these hormones in individual magnocellular neurosecretory cells. Both oxytocin-and vasopressin-producing magnocellular neurosecretory cells displayed Na V 1.7 immunolabeling (Figure 1). Approximately 72% (33 of 46) of oxytocin-producing and 53% (59 of 112) of vasopressin-producing MSC expressed Na V 1.7 labeling above background levels.
Salt-loading induced a substantial increase in the level of Na V 1.7 immunoreactivity in magnocellular neurosecretory cells of the supraoptic nucleus compared to magnocellular neurosecretory cells in control rats ( Figure 2A). In addition to the detection of greater numbers of magnocellular neurosecretory cells that displayed Na V 1.7 immunolabeling, the intensity of Na V 1.7 signal Figure 1 Na v 1.7 expression in vasopressin-and oxytocinproducing magnocellular neurosecretory cells in supraoptic nucleus. Magnocellular neurosecretory neurons (MSN) of the supraoptic nucleus (SON) exhibit robust vasopressin and oxytocin immunolabeling (green). MSN of the SON display Na v 1.7 immunoreactivity (red). Double-immunocytochemical studies with antibodies to vasopressin or oxytocin and Na v 1.7 demonstrate that both peptide-producing cell-types exhibit co-localization (magenta) with Na v 1.7. Merged image of vasopressin or oxytocin with Na v 1.7 is presented as magenta to enhance visualization of co-localization.
in some magnocellular neurosecretory neurons was markedly greater than that observed in magnocellular neurosecretory cells from control rats. Quantification of the mean intensity of Na V 1.7 signal within the circumscribed supraoptic nucleus demonstrated a significant up regulation of Na V 1.7 in response to salt-loading challenge ( Figure 2B). These observations demonstrate that, in addition to up regulation of the TTX-S sodium channels Na V 1.2 and Na V 1.6 within magnocellular neurosecretory cells of the supraoptic nucleus with salt-loading, the level of Na V 1.7 protein in these cells is significantly increased in osmotically-challenged rats.

Discussion
In this study we have demonstrated that Na V 1.7 is present within neurons within the hypothalamic supraoptic nucleus, specifically within vasopressin-and oxytocin-producing magnocellular neurosecretory neurons. We also show that the level of Na V 1.7 protein in these cells is not fixed but, on the contrary, is dynamic, increasing as a result of salt-loading.
A role Na V 1.7 in electrogenesis in DRG neurons is well-established, and it is clear that Na V 1.7 functions as a threshold channel in these neurons, amplifying small depolarizing inputs to bring the cell to threshold for action potential generation [26,27] and possibly facilitating invasion into, and/or transmitter release from, preterminal axons within the spinal cord dorsal horn [1,28]. In contrast, a functional role of Na V 1.7 within supraoptic neurons is less well understood. Action potential bursts, triggered by osmotic changes, lead to release of vasopressin by supraoptic magnocellular neurons [19] and it is known that tetrodotoxin-sensitive sodium channels contribute to this bursting [20][21][22].
Supraoptic magnocellular neurons are known to be highly dynamic. It is known that, in response to changes in osmolality, the expression of peptides within these cells changes, and they change in size [29]. In parallel, it has been shown that in response to increased osmolarity there are changes in deployment of sodium channels, with up-regulated expression of the Na V 1.2 and Na V 1.6 alpha subunits, and of the sodium channel beta-1 and beta-2 subunits [24,25]. The present results show that the level of Na V 1.7 protein, like that of Na V 1.2 and Na V 1.6 [24,25], is dynamic, and is up-regulated within supraoptic magnocellular neurons exposed to osmotic stress via salt-loading. A previous study [24] demonstrated an increase in the amplitude of the transient Na + current, and an even greater increase in the amplitude and density of the Na + currents evoked by slow ramp stimuli in supraoptic neurons following salt-loading. While definitive identification of the current as Na V 1.7 current would require specific blockade or knockout, both of these types of current have been observed to be produced by Na V 1.7 [26]. Because Na V 1.7 is present within vasopressin neurons, it seems likely that this sodium channel isoform plays some role in vasopressin release in response to the osmotic stress imposed by salt-loading.
Although only low levels of Na V 1.7 have been reported in the hypothalamus in primates [13], it is possible that the density of Na V 1.7 channels within magnocellular neurons of the human supraoptic nucleus, like that in rodents, is subject to up-regulation in response to some forms of stress. Na V 1.7 blockers are currently under development as potential pharmacotherapeutics for pain [30][31][32][33][34]. Hypothalamic dysfunction has not been observed thus far in families with channelopathyassociated insensitivity to pain due to null mutations in the gene encoding Na V 1.7. However, functional Na V 1.7 channels are absent beginning in early embryogenesis in affected individuals in these families, and the possibility that there might be compensatory changes in hypothalamic neurons which maintain relatively normal function in these cells cannot be excluded. Whether levels of Na V 1.7 are increased in response to environmental factors or stress within the human hypothalamus, and whether Na V 1.7 plays a functional role in hypothalamic neurons in humans, is not known. Until these questions are resolved, the present findings suggest the need for assessment of hypothalamic function in patients carrying Na V 1.7 mutations especially when subjected to stress, and for monitoring of hypothalamic function as Na V 1.7 blocking agents are studied.

Conclusions
In summary, our results demonstrate that sodium channel Na V 1.7 is expressed in vasopressin-producing and oxytocin-producing magnocellular neurosecretory neurons of the rat hypothalamic supraoptic nucleus. The level of Na V 1.7 immunoreactivity in the supraoptic nucleus is significantly increased following salt-loading, suggesting a contribution of Na V 1.7 in the response of magnocellular neurosecretory neurons to osmotic stress. While it is not yet known whether levels of expression of Na V 1.7 are increased in response to environmental factors or stress within the human hypothalamus, or whether Na V 1.7 plays a functional role in hypothalamic neurons in humans, the present findings suggest the need for assessment of hypothalamic function in patients carrying Na V 1.7 mutations, especially when subjected to stress, and for monitoring of hypothalamic function as Na V 1.7 blocking agents are studied.

Salt loading
Adult male Sprague-Dawley rats (200-220 g), housed under a 12 h-12 h dark-light cycle, were salt-loaded with 2% NaCl (ad libitum) in their drinking water and unlimited access to food. Rats were sacrificed for immunocytochemical investigation 7 days following salt loading. All experiments were approved by the VA Connecticut Healthcare System Institutional Animal Care and Use Committee. To confirm the extent of salt loading, plasma osmotic pressure of the rats was measured (vapor pressure osmometer model 5500, Wescor, USA). Body weights were significantly (p < .05) lower in saltloaded (186.6 ± 4.7 g) compared to control (248.6 ± 1.9) rats. Six control and 6 salt-loaded rats were used for the immunocytochemistry studies.

Quantification
Tissue sections were examined with a Nikon C1 confocal microscope (Nikon USA, Melville, NY), using a 20× objective and operating in single mode for detection of Na V 1.7 alone or in frame lamba (sequential) mode for detection of Na V 1.7 and vasopressin or oxytocin to prevent possible bleed-through between 488 and Cy3 channels.
For detection of Na V 1.7 in the supraoptic nucleus (SON), images were acquired from 6 control and 6 saltloaded rats, utilizing the same confocal settings for acquisition of Na V 1.7 immunofluorescent signals. Images were opened in Nikon Elements and the mean signal intensity of the circumscribed SON was calculated by the software.
For co-localization of Na V 1.7 and vasopressin or oxytocin in SON neurons, signals for Na V 1.7 and vasopressin were thresholded at intensities 20% above background levels, and the percentage of vasopressin neurons expressing Na V 1.7 was calculated. Data are presented as mean ± SEM and statistical analysis was performed with Excel Student's t-test, with p < 0.05 considered significant.