BACKGROUND
the rodent taste bud is a collection of approximately 50– <dig> spindle-shaped cells, termed taste cells.
a typical taste bud extends from the basal lamina to the taste pore, where apical microvilli extend into the oral cavity and interact with sapid molecules  <cit> .
murine taste buds contain three primary differentiated cell types: type i , type ii  and type iii  cells .
type i cells are the most abundant cell type and are characterized by the presence of prominent invaginations in the electron-dense nuclei and membrane-bound granules in the apical cytoplasm.
type i cells possess several long, slender microvilli that extend into the taste pore.
type i cells have sheet-like membrane extensions that envelop other cells in a glia-like manner and have been reported to express blood group antigen h  <cit> .
type i cells may be responsible for the “pore substance” released into the taste pore  <cit> .
the ecto-atpase, ntpdase <dig>  is present on the plasma membranes of type i cells  <cit> , which may function in the degradation of atp  <cit> .
vandenbeuch et al.
<cit>  have reported the presence of amiloride-sensitive na+ channels in type i cells.
type ii  cells have ovoid to circular nuclear profiles, electron-lucent cytoplasm and prominent smooth endoplasmic reticulum in the supranuclear cytoplasm.
type ii cells possess short, brush-like microvilli of the same length and express the immunocytochemical markers ip3r <dig>  <cit>  and plcβ <dig>  <cit> .
the g-protein, α-gustducin, is also present in a subset of type ii cells  <cit> .
there are two non-overlapping subsets of type ii cells.
one subset expresses the g protein, a-gustducin, while the other subset expresses protein gene product  <dig>  .
type ii cells express combinations of taste receptor proteins for detecting sweet , bitter  and umami .
type ii cells that signal sweet taste express gα <dig>  <cit> .
although type ii cells detect bitter, sweet, and umami, they do not form classical synapses onto nerve fibers  <cit> .
type iii  cells are intermediate in morphology between type i and type ii cells.
type iii cell cytoplasm is electron-lucent like a type ii cell but the nuclei are slender with prominent invaginations like type i cells.
a type iii cell possesses a single, large, blunt, apical microvillus.
type iii cells also possess numerous, small clear vesicles and a variable amount of large dense-cored vesicles associated with classical synapses onto nerve processes.
murray  <cit>  proposed that the type iii cell was the gustatory receptor cell.
the type iii cell has been termed the output or presynaptic cell in taste buds  <cit> .
rodent type iii cells display immunoreactivity to neural cell adhesion molecule , serotonin  and the synaptic proteins snap- <dig>  synaptobrevin- <dig>  syntaxin- <dig>  and synaptophysin  <cit> .
non-overlapping subsets of type iii cells display immunoreactivity to serotonin or protein gene product  <dig>   <cit> .
recent studies indicate that type iii cells release 5-ht upon gustatory stimulation  <cit> .
the candidate sour taste receptor, pkd2l <dig>  is present in taste buds  <cit>  and is expressed by type iii taste cells  <cit> .
our laboratory has shown that classical synapses and the synaptic snare proteins snap- <dig>  synaptobrevin- <dig> and syntaxin- <dig> are associated with type iii cells  <cit> .
no classical synapses have been found associated with type ii cells.
nevertheless, it is generally accepted that type ii cells transduce bitter, sweet and umami  <cit> .
a recent major finding in taste signal transduction provides evidence that atp serves as a neurotransmitter released from taste cells to nerve processes in taste buds  <cit> .
extracellular atp signaling acts through two families of receptors: p2x ionotropic ligand-gated ion channel receptors  and p2y metabotropic ligand-gated ion channel receptors   <cit> .
evidence supports a major role for p2x <dig> and p2x <dig> subunits in mediating the transduction of atp  <cit> .
taste nerves express the ionotropic atp-gated p2x <dig> and p2x <dig> receptors in rodent taste buds  <cit> .
atp was first shown to be a neurotransmitter in rodent taste buds when finger et al.
<cit>  showed that atp was released in response to gustatory stimuli.
in addition, they showed that responses to bitter, sweet and umami stimuli were eliminated in p2x2/p2x3dbl−/− mice.
p2x2/p2x3dbl−/− mice have also been shown not to taste nacl or the artificial sweetener sc <dig>  <cit> .
residual chemosensing abilities to detect bitter and sweet stimuli are believed to be mediated by postingestive detection  <cit> .
kataoka et al.
<cit>  observed expression of p2x <dig> receptors in intragemmal nerve processes of the rat.
ishida et al.
<cit>  found that p2x2- and p2x3-lir fibers in fungiform papillae originate from the chorda tympani nerve.
p2x <dig> and p2x <dig> receptors are also present in rat trigeminal ganglion neurons  <cit> .
recently, hayato et al.
<cit>  reported that p2x <dig> and p2x <dig> receptors are expressed on type ii and type iii taste bud cells of the mouse.
studies describing the non-vesicular release of atp within taste buds were published by huang et al.
<cit>  and romanov et al.
<cit> .
romanov et al.
<cit>  proposed that a non-vesicular release of atp occurs through connexin- <dig> hemichannels, while huang et al.
<cit>  proposed that atp is released through pannexin- <dig> hemichannels.
huang et al.
<cit>  have also proposed autocrine and paracrine roles for atp and serotonin in mouse taste buds.
release of atp from type ii cells onto type iii cells is thought to elicit the release of serotonin by type iii cells.
this serotonin release then provides negative feedback onto the type ii cells, resulting in decreased release of atp.
in the present study we have used immunofluorescence and immunoelectron microscopy to demonstrate that: 1) most all intragemmal nerve processes display p2x2-like immunoreactivity , 2) type iii cells form conventional synaptic contacts onto p2x2-lir nerve processes, 3) p2x <dig> receptors are distributed in a punctate manner adjacent to contacts with type ii cells, 4) large, atypical mitochondria with tubular cristae are found specifically in type ii cells at sites of close apposition with nerve processes.
RESULTS
p2x2-lir is present in intragemmal nerve processes in rat circumvallate taste buds .
the p2x <dig> immunoreactive nerve processes ramify throughout the entire taste bud, extending apically from the basal lamina almost to the taste pore.
intense immunoreactivity can also be seen in the subgemmal nerve plexuses located below the basal lamina.
however, p2x2-lir was not observed in the non-gustatory lingual epithelium surrounding taste buds.
p2x <dig> and ip3r3
ip3r <dig> immunoreactivity is commonly used as a marker for type ii cells.
ip3r3-lir cells have large, circular nuclei, a distinguishing feature of type ii cells .
the immunoreactivity is cytoplasmic only; nuclei display no staining.
cytoplasmic immunoreactivity extends evenly throughout the cytoplasm from the basal lamina to the taste pore.
p2x2-lir nerve processes are often in close apposition with ip3r3-lir cells.
some of these contacts are in the form of discrete terminal puncta, as shown in figure 1c .
this particular cell is contacted just apical to the nuclear region by two small nerve processes that end as swollen terminals.
cell # <dig>  has a large surface in contact with a nerve process.
at the other extreme, some ip3r3-lir cells are virtually enveloped by p2x2-lir nerve processes .
p2x <dig> and syntaxin-1
syntaxin- <dig>  the t-snare protein, has been demonstrated to be a cell marker for type iii cells as well as nerve processes  <cit> .
double labeling of p2x <dig>  with syntaxin- <dig>  shows syntaxin-1-lir is present in the type iii taste cells and both subgemmal and intragemmal nerve processes.
all syntaxin-1-lir nerve processes also display p2x2-lir and vice versa.
we did not find any syntaxin-1-lir taste cells expressing p2x2-lir , but the cells immunoreactive for syntaxin- <dig> are in close contact with p2x2-lir nerve processes, suggesting that p2x2-lir nerve processes are in close contact with type iii cells.
p2x <dig> and serotonin
experiments using p2x <dig>  and 5-ht , a known cell marker to be present in a subset of type iii cells  <cit> , were performed to determine if p2x2-lir nerve processes contact this subset of type iii cells.
5-ht-lir  is present in a small subset of taste cells  <cit> .
serotonin -lir cells are narrow and fusiform shaped, typical of type iii cells.
immunoreactivity in the cytoplasm extends from the basal lamina to the taste pore, and is present only in taste cells.
5-ht–lir cells resemble syntaxin-1-lir cells in both shape and structure, and have been shown to colocalize with a subset of syntaxin-1-lir  cells.
no colocalization occurs between 5-ht-lir and p2x2-lir , and no 5-ht immunoreactivity is present in the nerve processes.
all nerve processes that are in close apposition with type iii cells display p2x2-lir .
p2x2-lir nerve processes
immunoelectron microscopy using diaminobenzidine  demonstrates that p2x <dig> is present in nerve processes .
all of the intragemmal nerve processes that we observed in rat circumvallate taste buds display p2x2-lir.
p2x2-lir nerve processes are in close apposition with both type ii and type iii taste cells.
type ii taste cells have numerous mitochondria found adjacent to swollen smooth endoplasmic reticulum.
these cells form close contacts with p2x2-lir nerve processes.
the taste cell shown in the inset for figure  <dig> contains electron-dense patches of heterochromatin in the nucleus, which is a distinguishing feature of type iii cells.
regular mitochondria are often found in abundance within the p2x2-lir nerve processes.
synapses
atypical mitochondria in type ii cells
no conventional synapses were observed from type ii taste cells onto nerve processes.
we did observe, however, that type ii cells often contained large, “atypical” mitochondria that were located only at close appositions between the membranes of type ii cells and p2x2-lir nerve processes .
the cristae of the atypical mitochondria exhibit a “twisted-energized” or “swollen twisted-energized configuration”  <cit> , resembling electron-dense sacs or tubules within the mitochondrion .
this contrasts with the typical mitochondria distributed throughout the cytoplasm of type ii taste cells, which are elongate, slender structures rarely exceeding  <dig>  μm in width and possessing lamellar or baffle cristae .
the “atypical” mitochondria that we have observed have diameters two to three times larger than conventional mitochondria.
the atypical mitochondria are separated from the cytoplasmic leaflet of the taste cell membrane by a gap of approximately  <dig> nm .
immunogold microscopy
our results indicate that p2x <dig> receptors are concentrated at the junctions between type ii cells and nerve processes in a punctate manner .
since  <dig> nm immunogold particles were used in this research, we took pictures with a magnification of  <dig> .
the immunogold particles are very small, hence we masked each gold particle with a transparent red dot using photoshop.
the signal is very specific, being located near the membrane of nerve processes in close apposition with type ii cells .
virtually no background immunoreactivity for p2x <dig> was observed.
no immunogold particles were found at the synapses between type iii cells and nerve processes.
discussion
p2x2/ <dig> receptors in intragemmal nerve processes were first reported by bo et al.
in  <dig>  <cit> .
finger et al.
<cit>  found that p2x2/ <dig> double knockout mice are not responsive to sweet, umami and bitter stimuli, and proposed that atp acts as a neurotransmitter in taste buds.
in a previous study  <cit> , we used conventional electron microscopy to compare taste bud ultrastructure in p2x2/ <dig> double knockout mice with taste buds in wild-type mice.
there were no differences at the ultrastructural level between p2x2/ <dig> double knockout mice and the wild-type mice taste buds.
taste cells and nerve processes in the p2x2/ <dig> double knockout mouse taste buds show normal ultrastructural features.
based on our present fluorescence and immunoelectron microscopical studies we believe that both perigemmal and most, if not all intragemmal nerve processes display p2x2-lir.
numerous immunoreactive nerve processes can be seen coursing throughout the lamina propria as well as several immunoreactive nerve processes that appear to be projecting from the subgemmal plexuses through the basal lamina into the taste buds.
all p2x2-lir nerve processes display syntaxin-1-lir.
our immunoelectron microscopical results show that the p2x2-lir nerve processes contain numerous mitochondria.
these nerve processes are in close apposition with type ii or type iii cells, and some immunoreactive nerve processes even envelop much of the taste cells.
using diaminobenzidine  immunoelectron microscopy, p2x2-lir appears to be distributed homogeneously throughout the nerve processes.
using colloidal gold immunoelectron microscopy, however, the gold particles were localized on the nerve process membranes at close appositions with the taste cell membranes.
type ii cells are in intimate contact with p2x2-lir intragemmal nerve processes, providing a pathway for functional connectivity between type ii cells and p2x2-lir nerve processes.
this is best shown in figure 1c, which shows an ip3r3-lir type ii cell  with contacts onto two expanded terminals of a p2x2-lir nerve process.
the results were similar when we colocalized p2x2-lir with the type iii cell markers, 5-ht  <cit>  and syntaxin- <dig>  <cit> .
although the type iii cells with 5-ht and/or syntaxin-1-lir did not display p2x2-lir, these type iii cells are in intimate contact with p2x <dig> immunoreactive nerve processes.
in the present study, we did not find taste cells in rat circumvallate taste buds displaying p2x2-lir.
however, hayato et al.,  <cit>  indicated that a subset of type iii taste cells in mouse fungiform taste buds express p2x2-lir.
based on our data from confocal microscopy, dab immunoelectron and immunogold microscopy, p2x <dig> is only present in nerve processes in rat circumvallate taste buds.
we did not find any taste cells displaying p2x2-lir.
although some p2x2-lir in figure 1a appears to be in a taste cell, the colocalization results in figure 1c demonstrate that the p2x2-lir was present in a nerve process that enveloped an ip3r3-lir taste cell .
another possibility is that these differences in results may arise from differences between rat and mouse taste buds.
we have previously observed that some antibodies show different immunoreactivity patterns in rat and mouse taste buds, or even different immunoreactivity patterns between different papillae of the same species  <cit> .
in our previous studies  <cit> , taste cell synapses were found to be associated only with type iii cells.
in the present study we found that all of the postsynaptic nerve processes that we observed displayed p2x2-lir.
it is significant that type iii cells form synaptic contacts containing small, clear vesicles and large, dense-cored vesicles onto p2x2-lir nerve processes.
in addition, our results indicates that type iii cells express pannexin-1-lir  <cit> .
thus, type iii cells may release transmitters from small, clear vesicles and/or large, dense-cored vesicles onto the p2x2-lir nerve fibers, and type iii cells might also release atp onto nerve processes and/or other taste cells.
most mitochondria in taste cells are long, slender structures rarely exceeding  <dig>  μm in diameter  <cit> .
the atypical mitochondria that we have observed have diameters two to three times larger than conventional mitochondria.
typically the cristae of the atypical mitochondria exhibit a “twisted-energized” or “swollen twisted-energized configuration”  <cit> , resembling electron-dense sacs or tubules within the mitochondrion.
the outer membranes of these atypical mitochondria are closely apposed to the cytoplasmic leaflet of the type ii taste cell’s membrane .
what are the roles of the atypical mitochondria in taste cell function?
based on their location at the sites of close apposition between type ii cells and nerve processes, we speculate that the atypical mitochondria may be the source of atp that is released through the pannexin/connexin hemichannels onto nerve processes.
approximately 94% of the atp synthase in a mitochondrion is located in the cristal membrane  <cit> .
increases in the ratio of cristal membrane to inner membrane is associated with increases in oxidative phosphorylation and the production of atp  <cit> .
because of their large size, atypical mitochondria at taste cell-nerve fiber contacts may contain significant amounts of atp.
thus the atypical mitochondria we have observed may be ideal sources of atp for use as a neurotransmitter at these contacts.
another possibility is that mitochondria may modulate the open/closed state of pannexin/connexin hemichannels by a regulated uptake and release of ca2+, which is known to modulate the coupling coefficient of gap junctions.
support for this idea comes from studies which have shown that mitochondria can take up and release ca2+ in response to gpcr activation via the ip <dig> pathway  <cit> .
the ip <dig> pathway is present in type ii taste cells  <cit>  and is required for bitter, sweet, and umami transduction  <cit> .
in addition, the opening of pannexin hemichannels has been reported to be controlled by ca2+ <cit> .
thus, whether the atypical mitochondria are the source of atp, regulate atp release through ca2+, or both, the close apposition of the unusually large mitochondria with pannexin hemichannels and nerve fibers containing p2x <dig> receptors provides a potential pathway for the control of atp release.
CONCLUSIONS
p2x2-like immunoreactivity is present in intragemmal nerve processes in rat circumvallate taste buds.
the immunoreactive nerves are in close contact with the ip3r3-lir type ii cells, syntaxin-1-lir and/or 5-ht-lir type iii cells.
taste cell synapses are observed only from type iii taste cells onto p2x2-lir nerve processes.
large, “atypical” mitochondria in the type ii taste cells are found only at close appositions with p2x2-lir nerve processes.
p2x <dig> immunogold particles are concentrated at the membranes of nerve processes at close appositions with taste cells.
based on our immunofluorescence and immunoelectron microscopical studies, we suggest that atp may be a key neurotransmitter in taste transduction, and type ii cells may release atp to activate the p2x <dig> receptors in nerve processes.

 the rodent taste bud is a collection of approximately 50– spindle-shaped cells, termed taste cells. a typical taste bud extends from the basal lamina to the taste pore, where apical microvilli extend into the oral cavity and interact with sapid molecules. murine taste buds contain three primary differentiated cell types: type i, type ii and type iii cells. type i cells are the most abundant cell type and are characterized by the presence of prominent invaginations in the electron-dense nuclei and membrane-bound granules in the apical cytoplasm. type i cells possess several long, slender microvilli that extend into the taste pore. type i cells have sheet-like membrane extensions that envelop other cells in a glia-like manner and have been reported to express blood group antigen h. type i cells may be responsible for the “pore substance” released into the taste pore. the ecto-atpase, ntpdase is present on the plasma membranes of type i cells, which may function in the degradation of atp. vandenbeuch et al. have reported the presence of amiloride-sensitive na+ channels in type i cells. type ii cells have ovoid to circular nuclear profiles, electron-lucent cytoplasm and prominent smooth endoplasmic reticulum in the supranuclear cytoplasm. type ii cells possess short, brush-like microvilli of the same length and express the immunocytochemical markers ip3r and plcβ. the g-protein, α-gustducin, is also present in a subset of type ii cells. there are two non-overlapping subsets of type ii cells. one subset expresses the g protein, a-gustducin, while the other subset expresses protein gene product. type ii cells express combinations of taste receptor proteins for detecting sweet, bitter and umami. type ii cells that signal sweet taste express gα. although type ii cells detect bitter, sweet, and umami, they do not form classical synapses onto nerve fibers. type iii cells are intermediate in morphology between type i and type ii cells. type iii cell cytoplasm is electron-lucent like a type ii cell but the nuclei are slender with prominent invaginations like type i cells. a type iii cell possesses a single, large, blunt, apical microvillus. type iii cells also possess numerous, small clear vesicles and a variable amount of large dense-cored vesicles associated with classical synapses onto nerve processes. murray proposed that the type iii cell was the gustatory receptor cell. the type iii cell has been termed the output or presynaptic cell in taste buds. rodent type iii cells display immunoreactivity to neural cell adhesion molecule, serotonin and the synaptic proteins snap- synaptobrevin- syntaxin- and synaptophysin. non-overlapping subsets of type iii cells display immunoreactivity to serotonin or protein gene product. recent studies indicate that type iii cells release 5-ht upon gustatory stimulation. the candidate sour taste receptor, pkd2l is present in taste buds and is expressed by type iii taste cells. our laboratory has shown that classical synapses and the synaptic snare proteins snap- synaptobrevin- and syntaxin- are associated with type iii cells. no classical synapses have been found associated with type ii cells. nevertheless, it is generally accepted that type ii cells transduce bitter, sweet and umami. a recent major finding in taste signal transduction provides evidence that atp serves as a neurotransmitter released from taste cells to nerve processes in taste buds. extracellular atp signaling acts through two families of receptors: p2x ionotropic ligand-gated ion channel receptors and p2y metabotropic ligand-gated ion channel receptors. evidence supports a major role for p2x and p2x subunits in mediating the transduction of atp. taste nerves express the ionotropic atp-gated p2x and p2x receptors in rodent taste buds. atp was first shown to be a neurotransmitter in rodent taste buds when finger et al. showed that atp was released in response to gustatory stimuli. in addition, they showed that responses to bitter, sweet and umami stimuli were eliminated in p2x2/p2x3dbl−/− mice. p2x2/p2x3dbl−/− mice have also been shown not to taste nacl or the artificial sweetener sc. residual chemosensing abilities to detect bitter and sweet stimuli are believed to be mediated by postingestive detection. kataoka et al. observed expression of p2x receptors in intragemmal nerve processes of the rat. ishida et al. found that p2x2- and p2x3-lir fibers in fungiform papillae originate from the chorda tympani nerve. p2x and p2x receptors are also present in rat trigeminal ganglion neurons. recently, hayato et al. reported that p2x and p2x receptors are expressed on type ii and type iii taste bud cells of the mouse. studies describing the non-vesicular release of atp within taste buds were published by huang et al. and romanov et al.. romanov et al. proposed that a non-vesicular release of atp occurs through connexin- hemichannels, while huang et al. proposed that atp is released through pannexin- hemichannels. huang et al. have also proposed autocrine and paracrine roles for atp and serotonin in mouse taste buds. release of atp from type ii cells onto type iii cells is thought to elicit the release of serotonin by type iii cells. this serotonin release then provides negative feedback onto the type ii cells, resulting in decreased release of atp. in the present study we have used immunofluorescence and immunoelectron microscopy to demonstrate that: 1) most all intragemmal nerve processes display p2x2-like immunoreactivity, 2) type iii cells form conventional synaptic contacts onto p2x2-lir nerve processes, 3) p2x receptors are distributed in a punctate manner adjacent to contacts with type ii cells, 4) large, atypical mitochondria with tubular cristae are found specifically in type ii cells at sites of close apposition with nerve processes.  p2x2-lir is present in intragemmal nerve processes in rat circumvallate taste buds. the p2x immunoreactive nerve processes ramify throughout the entire taste bud, extending apically from the basal lamina almost to the taste pore. intense immunoreactivity can also be seen in the subgemmal nerve plexuses located below the basal lamina. however, p2x2-lir was not observed in the non-gustatory lingual epithelium surrounding taste buds. p2x and ip3r3 ip3r immunoreactivity is commonly used as a marker for type ii cells. ip3r3-lir cells have large, circular nuclei, a distinguishing feature of type ii cells. the immunoreactivity is cytoplasmic only; nuclei display no staining. cytoplasmic immunoreactivity extends evenly throughout the cytoplasm from the basal lamina to the taste pore. p2x2-lir nerve processes are often in close apposition with ip3r3-lir cells. some of these contacts are in the form of discrete terminal puncta, as shown in figure 1c. this particular cell is contacted just apical to the nuclear region by two small nerve processes that end as swollen terminals. cell # has a large surface in contact with a nerve process. at the other extreme, some ip3r3-lir cells are virtually enveloped by p2x2-lir nerve processes. p2x and syntaxin-1 syntaxin- the t-snare protein, has been demonstrated to be a cell marker for type iii cells as well as nerve processes. double labeling of p2x with syntaxin- shows syntaxin-1-lir is present in the type iii taste cells and both subgemmal and intragemmal nerve processes. all syntaxin-1-lir nerve processes also display p2x2-lir and vice versa. we did not find any syntaxin-1-lir taste cells expressing p2x2-lir, but the cells immunoreactive for syntaxin- are in close contact with p2x2-lir nerve processes, suggesting that p2x2-lir nerve processes are in close contact with type iii cells. p2x and serotonin experiments using p2x and 5-ht, a known cell marker to be present in a subset of type iii cells, were performed to determine if p2x2-lir nerve processes contact this subset of type iii cells. 5-ht-lir is present in a small subset of taste cells. serotonin -lir cells are narrow and fusiform shaped, typical of type iii cells. immunoreactivity in the cytoplasm extends from the basal lamina to the taste pore, and is present only in taste cells. 5-ht–lir cells resemble syntaxin-1-lir cells in both shape and structure, and have been shown to colocalize with a subset of syntaxin-1-lir cells. no colocalization occurs between 5-ht-lir and p2x2-lir, and no 5-ht immunoreactivity is present in the nerve processes. all nerve processes that are in close apposition with type iii cells display p2x2-lir. p2x2-lir nerve processes immunoelectron microscopy using diaminobenzidine demonstrates that p2x is present in nerve processes. all of the intragemmal nerve processes that we observed in rat circumvallate taste buds display p2x2-lir. p2x2-lir nerve processes are in close apposition with both type ii and type iii taste cells. type ii taste cells have numerous mitochondria found adjacent to swollen smooth endoplasmic reticulum. these cells form close contacts with p2x2-lir nerve processes. the taste cell shown in the inset for figure contains electron-dense patches of heterochromatin in the nucleus, which is a distinguishing feature of type iii cells. regular mitochondria are often found in abundance within the p2x2-lir nerve processes. synapses atypical mitochondria in type ii cells no conventional synapses were observed from type ii taste cells onto nerve processes. we did observe, however, that type ii cells often contained large, “atypical” mitochondria that were located only at close appositions between the membranes of type ii cells and p2x2-lir nerve processes. the cristae of the atypical mitochondria exhibit a “twisted-energized” or “swollen twisted-energized configuration”, resembling electron-dense sacs or tubules within the mitochondrion. this contrasts with the typical mitochondria distributed throughout the cytoplasm of type ii taste cells, which are elongate, slender structures rarely exceeding μm in width and possessing lamellar or baffle cristae. the “atypical” mitochondria that we have observed have diameters two to three times larger than conventional mitochondria. the atypical mitochondria are separated from the cytoplasmic leaflet of the taste cell membrane by a gap of approximately nm. immunogold microscopy our results indicate that p2x receptors are concentrated at the junctions between type ii cells and nerve processes in a punctate manner. since nm immunogold particles were used in this research, we took pictures with a magnification of. the immunogold particles are very small, hence we masked each gold particle with a transparent red dot using photoshop. the signal is very specific, being located near the membrane of nerve processes in close apposition with type ii cells. virtually no background immunoreactivity for p2x was observed. no immunogold particles were found at the synapses between type iii cells and nerve processes. discussion p2x2/ receptors in intragemmal nerve processes were first reported by bo et al. in. finger et al. found that p2x2/ double knockout mice are not responsive to sweet, umami and bitter stimuli, and proposed that atp acts as a neurotransmitter in taste buds. in a previous study, we used conventional electron microscopy to compare taste bud ultrastructure in p2x2/ double knockout mice with taste buds in wild-type mice. there were no differences at the ultrastructural level between p2x2/ double knockout mice and the wild-type mice taste buds. taste cells and nerve processes in the p2x2/ double knockout mouse taste buds show normal ultrastructural features. based on our present fluorescence and immunoelectron microscopical studies we believe that both perigemmal and most, if not all intragemmal nerve processes display p2x2-lir. numerous immunoreactive nerve processes can be seen coursing throughout the lamina propria as well as several immunoreactive nerve processes that appear to be projecting from the subgemmal plexuses through the basal lamina into the taste buds. all p2x2-lir nerve processes display syntaxin-1-lir. our immunoelectron microscopical results show that the p2x2-lir nerve processes contain numerous mitochondria. these nerve processes are in close apposition with type ii or type iii cells, and some immunoreactive nerve processes even envelop much of the taste cells. using diaminobenzidine immunoelectron microscopy, p2x2-lir appears to be distributed homogeneously throughout the nerve processes. using colloidal gold immunoelectron microscopy, however, the gold particles were localized on the nerve process membranes at close appositions with the taste cell membranes. type ii cells are in intimate contact with p2x2-lir intragemmal nerve processes, providing a pathway for functional connectivity between type ii cells and p2x2-lir nerve processes. this is best shown in figure 1c, which shows an ip3r3-lir type ii cell with contacts onto two expanded terminals of a p2x2-lir nerve process. the results were similar when we colocalized p2x2-lir with the type iii cell markers, 5-ht and syntaxin-. although the type iii cells with 5-ht and/or syntaxin-1-lir did not display p2x2-lir, these type iii cells are in intimate contact with p2x immunoreactive nerve processes. in the present study, we did not find taste cells in rat circumvallate taste buds displaying p2x2-lir. however, hayato et al., indicated that a subset of type iii taste cells in mouse fungiform taste buds express p2x2-lir. based on our data from confocal microscopy, dab immunoelectron and immunogold microscopy, p2x is only present in nerve processes in rat circumvallate taste buds. we did not find any taste cells displaying p2x2-lir. although some p2x2-lir in figure 1a appears to be in a taste cell, the colocalization results in figure 1c demonstrate that the p2x2-lir was present in a nerve process that enveloped an ip3r3-lir taste cell. another possibility is that these differences in results may arise from differences between rat and mouse taste buds. we have previously observed that some antibodies show different immunoreactivity patterns in rat and mouse taste buds, or even different immunoreactivity patterns between different papillae of the same species. in our previous studies, taste cell synapses were found to be associated only with type iii cells. in the present study we found that all of the postsynaptic nerve processes that we observed displayed p2x2-lir. it is significant that type iii cells form synaptic contacts containing small, clear vesicles and large, dense-cored vesicles onto p2x2-lir nerve processes. in addition, our results indicates that type iii cells express pannexin-1-lir. thus, type iii cells may release transmitters from small, clear vesicles and/or large, dense-cored vesicles onto the p2x2-lir nerve fibers, and type iii cells might also release atp onto nerve processes and/or other taste cells. most mitochondria in taste cells are long, slender structures rarely exceeding μm in diameter. the atypical mitochondria that we have observed have diameters two to three times larger than conventional mitochondria. typically the cristae of the atypical mitochondria exhibit a “twisted-energized” or “swollen twisted-energized configuration”, resembling electron-dense sacs or tubules within the mitochondrion. the outer membranes of these atypical mitochondria are closely apposed to the cytoplasmic leaflet of the type ii taste cell’s membrane. what are the roles of the atypical mitochondria in taste cell function? based on their location at the sites of close apposition between type ii cells and nerve processes, we speculate that the atypical mitochondria may be the source of atp that is released through the pannexin/connexin hemichannels onto nerve processes. approximately 94% of the atp synthase in a mitochondrion is located in the cristal membrane. increases in the ratio of cristal membrane to inner membrane is associated with increases in oxidative phosphorylation and the production of atp. because of their large size, atypical mitochondria at taste cell-nerve fiber contacts may contain significant amounts of atp. thus the atypical mitochondria we have observed may be ideal sources of atp for use as a neurotransmitter at these contacts. another possibility is that mitochondria may modulate the open/closed state of pannexin/connexin hemichannels by a regulated uptake and release of ca2+, which is known to modulate the coupling coefficient of gap junctions. support for this idea comes from studies which have shown that mitochondria can take up and release ca2+ in response to gpcr activation via the ip pathway. the ip pathway is present in type ii taste cells and is required for bitter, sweet, and umami transduction. in addition, the opening of pannexin hemichannels has been reported to be controlled by ca2+. thus, whether the atypical mitochondria are the source of atp, regulate atp release through ca2+, or both, the close apposition of the unusually large mitochondria with pannexin hemichannels and nerve fibers containing p2x receptors provides a potential pathway for the control of atp release.  p2x2-like immunoreactivity is present in intragemmal nerve processes in rat circumvallate taste buds. the immunoreactive nerves are in close contact with the ip3r3-lir type ii cells, syntaxin-1-lir and/or 5-ht-lir type iii cells. taste cell synapses are observed only from type iii taste cells onto p2x2-lir nerve processes. large, “atypical” mitochondria in the type ii taste cells are found only at close appositions with p2x2-lir nerve processes. p2x immunogold particles are concentrated at the membranes of nerve processes at close appositions with taste cells. based on our immunofluorescence and immunoelectron microscopical studies, we suggest that atp may be a key neurotransmitter in taste transduction, and type ii cells may release atp to activate the p2x receptors in nerve processes. 
the goal of the present study is to learn more about the nature of purinergic contacts in rat circumvallate taste buds by examining immunoreactivity to antisera directed against the purinergic receptor p2x p2x2-like immunoreactivity is present in intragemmal nerve processes in rat circumvallate taste buds. p2x2/p2x3dbl−/− mice are not responsive to sweet, umami and bitter stimuli, and it has been proposed that atp acts as a neurotransmitter in taste buds. intense immunoreactivity can also be seen in the subgemmal nerve plexuses located below the basal lamina. this supports the hypothesis that atp may be a key neurotransmitter in taste transduction and that type ii cells release atp, activating p2x receptors in nerve processes. taste cell synapses are observed only from type iii taste cells onto p2x2-lir nerve processes. taste nerve processes express the ionotropic purinergic receptors. the p2x immunoreactive nerve processes also display syntaxin-1-lir. unusually large, “atypical” mitochondria in the type ii taste cells are found only at close appositions with p2x2-lir nerve processes. p2x immunogold particles are concentrated at the membranes of nerve processes at close appositions with taste cells. based on our immunofluorescence and immunoelectron microscopical studies we believe that both perigemmal and most all intragemmal nerve processes display p2x2-lir. moreover, colloidal gold immunoelectron microscopy indicates that p2x2-lir in nerve processes is concentrated at sites of close apposition with type ii cells. recent studies indicate that the ionotropic purinergic receptors p2x2/p2x are present in rodent taste buds. yet it is generally accepted that type ii cells transduce bitter, sweet and umami stimuli. no classical synapses, however, have been found associated with type ii cells. the immunoreactive nerves are in close contact with the ip3r3-lir type ii cells and syntaxin-1-lir and/or 5-ht-lir type iii cells. our laboratory has shown that classical synapses and synaptic proteins are associated with type iii cells.
 our laboratory has shown that classical synapses and synaptic proteins are associated with type iii cells. yet it is generally accepted that type ii cells transduce bitter, sweet and umami stimuli. no classical synapses, however, have been found associated with type ii cells. recent studies indicate that the ionotropic purinergic receptors p2x2/p2x are present in rodent taste buds. taste nerve processes express the ionotropic purinergic receptors. p2x2/p2x3dbl−/− mice are not responsive to sweet, umami and bitter stimuli, and it has been proposed that atp acts as a neurotransmitter in taste buds. the goal of the present study is to learn more about the nature of purinergic contacts in rat circumvallate taste buds by examining immunoreactivity to antisera directed against the purinergic receptor p2x  p2x2-like immunoreactivity is present in intragemmal nerve processes in rat circumvallate taste buds. intense immunoreactivity can also be seen in the subgemmal nerve plexuses located below the basal lamina. the p2x immunoreactive nerve processes also display syntaxin-1-lir. the immunoreactive nerves are in close contact with the ip3r3-lir type ii cells and syntaxin-1-lir and/or 5-ht-lir type iii cells. taste cell synapses are observed only from type iii taste cells onto p2x2-lir nerve processes. unusually large, “atypical” mitochondria in the type ii taste cells are found only at close appositions with p2x2-lir nerve processes. p2x immunogold particles are concentrated at the membranes of nerve processes at close appositions with taste cells.  based on our immunofluorescence and immunoelectron microscopical studies we believe that both perigemmal and most all intragemmal nerve processes display p2x2-lir. moreover, colloidal gold immunoelectron microscopy indicates that p2x2-lir in nerve processes is concentrated at sites of close apposition with type ii cells. this supports the hypothesis that atp may be a key neurotransmitter in taste transduction and that type ii cells release atp, activating p2x receptors in nerve processes. 