Studies on the coexistence of substance P with other putative transmitters in the nodose and petrosal ganglia.

Visceral afferent neurons of the nodose and petrosal ganglia are immunoreactive (ir) for many neurotransmitters [e.g., substance P (SP), neurokinin A (NKA), calcitonin gene-related peptide (CGRP), and dopamine (tyrosine hydroxylase-ir; TH)]. Coexistence of SP-ir with NKA-, CGRP-, or TH-ir was studied in individual neurons of the rat ganglia using fluorescence immunocytochemistry. SP- and NKA-ir were present in equal numbers of cells and were consistently colocalized. SP- and CGRP-ir were found to be similarly distributed in scattered cells, concentrated mostly in the rostral pole of the nodose ganglion and in the petrosal ganglion. SP-ir completely coexisted with CGRP-ir. However, there was at least twice the number of CGRP-ir neurons as SP-ir neurons, and thus CGRP-ir neurons that did not contain SP-ir were also present. In contrast, SP- and TH-ir had different distributions in both the nodose and the petrosal ganglia. SP-ir was located in the more rostral regions of both the nodose and petrosal ganglia, whereas TH-ir was detected throughout the entire nodose ganglion and only in the most caudal region of the petrosal ganglion. There was no coexistence of SP- and TH-ir. These data demonstrate the differential localization and coexistence of putative transmitters in visceral sensory neurons in the nodose and petrosal ganglia.     

Axotomy alters putative neurotransmitters in visceral sensory neurons of the nodose and petrosal ganglia.

Acute peripheral axotomy of the visceral sensory neurons of the vagus and glossopharyngeal nerves removes peripheral depolarizing and trophic influences to their sensory ganglia. To study axotomy-induced changes in the putative neurotransmitters of visceral sensory neurons, rats were sacrificed 1, 3, 7 or 14 days after transection of either the cervical vagus and superior laryngeal nerves (to affect peripheral axotomy of the nodose ganglion) or the glossopharyngeal and carotid sinus nerves (to affect peripheral axotomy of the petrosal ganglion). The numbers of tyrosine hydroxylase (TH)-immunoreactive (ir), vasoactive intestinal peptide (VIP)-ir, calcitonin-gene-related peptide (CGRP)-ir, and substance P (SP)-ir neurons in the respective ganglia were analyzed in axotomized and control ganglia. In the nodose ganglion, axotomy of the cervical vagus resulted in a rapid (by 1 day) reduction in the number of TH-ir cells, whereas VIP-ir neurons were dramatically increased in number by 3 days. CGRP- and SP-ir cells in the nodose ganglion were relatively unaffected by axotomy. In the petrosal ganglion, axotomy of the glossopharyngeal and carotid sinus nerves greatly reduced the number of TH-ir cells but did not alter the number VIP-ir neurons. CGRP- and SP-ir neurons in the petrosal ganglion were reduced in number by axotomy. Thus, axotomy of visceral sensory neurons differentially changed the content and perhaps the expression of putative transmitters. Differential changes were seen among transmitters in a single ganglia and between ganglia. These data demonstrate the plasticity of putative neurotransmitter systems in visceral afferent systems of adult rats.     

Coexistence of S100β and putative transmitter agents in vagal and glossopharyngeal sensory neurons of the rat

The coexistence of S100β with calcitonin gene-related peptide (CGRP), substance P (SP), somatostatin (SOM), nicotinamide adenosine dinucleotide phosphate-diaphorase (NADPH-d), and tyrosine hydroxylase (TH) was examined in the glossopharyngeal and vagal sensory ganglia. S100β immunoreactive (-ir) neurons in the jugular and petrosal ganglia frequently colocalized CGRP- or SP-ir, whereas S100β-ir neurons in the nodose ganglion infrequently contained CGRP- or SP-ir. No S100β-ir neurons in the jugular and petrosal ganglia showed SOM-ir while the small number of SOM-ir neurons in the nodose ganglion colocalized S100β-ir. Many neurons in the nodose ganglion colocalized S100β-ir and NADPH-d activity, whereas S100β-ir neurons in the jugular and nodose ganglia infrequently contained NADPH-d activity. S100β- and TH-ir were frequently colocalized in nodose ganglion but not in petrosal or jugular ganglion neurons. These findings suggest relationships between S100β and specific putative transmitters in functions of subpopulations of vagal and glossopharyngeal sensory neurons.     

The coexistence of TrkA with putative transmitter agents and calcium-binding proteins in the vagal and glossopharyngeal sensory neurons of the adult rat

The presence of the neurotrophin receptor, TrkA, in neurochemically identified vagal and glossopharyngeal sensory neurons of the adult rat was examined. TrkA was colocalized with calcitonin gene-related peptide (CGRP), parvalbumin, or calbindin D-28k in neurons of the nodose, petrosal and/or jugular ganglia. In contrast, no TrkA-immunoreactive (ir) neurons in these ganglia colocalized tyrosine hydroxylase-ir. About one-half of the TrkA-ir neurons in the jugular and petrosal ganglia contained CGRP-ir, whereas only a few of the numerous TrkA-ir neurons in the nodose ganglion contained CGRP-ir. Although 43% of the TrkA-ir neurons in the nodose ganglion contained calbindin D-28k-ir, few or no TrkA-ir neurons in the petrosal or jugular ganglia were also labeled for either calcium-binding protein. These data show distinct colocalizations of TrkA with specific neurochemicals in vagal and glossopharyngeal sensory neurons, and suggest that nerve growth factor (NGF), the neurotrophin ligand for TrkA, plays a role in functions of specific neurochemically defined subpopulations of mature vagal and glossopharyngeal sensory neurons.     

Neurotrophins alter the numbers of neurotransmitter-ir mature vagal/glossopharyngeal visceral afferent neurons in vitro

Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10–100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.     

Neurotrophins alter the numbers of neurotransmitter-ir mature vagal/glossopharyngeal visceral afferent neurons in vitro

Mature nodose and petrosal ganglia neurons (placodally derived afferent neurons of the vagal and glossopharyngeal nerves) contain TrkA and TrkC, and transport specific neurotrophins [nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4)]. This study evaluated neurotrophin influences on the presence of neuropeptides and/or neurotransmitter enzymes in these visceral sensory neurons. NGF, NT-3 and NT-4 (10-100 ng/ml) were applied (5 days) to dissociated, enriched, cultures of mature nodose/petrosal ganglia neurons, and the neurons processed for tyrosine hydroxylase (TH), vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP) and neurofilament (NF-200) immunocytochemistry. Addition of NGF to nodose/petrosal ganglia neuron-enriched cultures significantly increased the number of TH-immunoreactive (ir) neurons, decreased the number of VIP-ir neurons in the cultures, and did not affect the numbers of CGRP-ir neurons. The addition of an NGF neutralizing antibody attenuated the effects of NGF on TH and VIP-ir neurons. NT-3 increased the number of VIP-ir neurons in the nodose/petrosal ganglia cultures and did not alter the numbers of TH-, or CGRP-ir neurons. The addition of an NT-3 neutralizing antibody attenuated the effects of NT-3 on VIP-ir neurons. NT-4 had no significant effects on the numbers of TH, VIP and CGRP-ir neurons. The absence of neurotrophin-induced changes in the numbers of NF-200-ir neurons in culture showed the lack of neurotrophin-mediated changes in survival of mature vagal afferent neurons. These data demonstrate that specific neurotrophins influence the numbers of neurons labeled for specific neurochemicals in nodose/petrosal ganglia cultures. These data, coupled with previous evidence for the presence of TrkA and TrkC mRNA and of the retrograde transport of NGF and NT-3, suggest important roles for NGF and NT-3 in the maintenance of transmitter phenotype of these mature visceral afferent neurons.     

Coexistence of calbindin D-28k and NADPH-diaphorase in vagal and glossopharyngeal sensory neurons of the rat

The presence and coexistence of calbindin D-28k-immunoreactivity (ir) and nicotinamide adenosine dinucleotide phosphate (NADPH)-diaphorase activity (a marker of neurons that are presumed to convert L-arginine to L-citrulline and nitric oxide) were examined in the glossopharyngeal and vagal sensory ganglia (jugular, petrosal and nodose ganglia) of the rat. Calbindin D-28k-ir nerve cells were found in moderate and large numbers in the petrosal and nodose ganglia, respectively. Some calbindin D-28k-ir nerve cells were also observed in the jugular ganglion. NADPH-diaphorase positive nerve cells were localized to the jugular and nodose ganglia and were rare in the petrosal ganglion. A considerable portion (33–51%) of the NADPH-diaphorase positive neurons in these ganglia colocalized calbindin D-28k-ir. The presence and colocalization of calbindin D-28k-ir and NADPH-diaphorase activity in neurotransmitter-identified subpopulations of visceral sensory neurons were also studied. In all three ganglia, calcitonin gene-related peptide (CGRP)-ir was present in many NADPH-diaphorase positive neurons, a subset of which also contained calbindin D-28k-ir. In the nodose ganglion, many (42%) of tyrosine hydroxylase (TH)-ir neurons also contained NADPH diaphorase activity but did not contain calbindin D-28k-ir. These data are consistent with a potential co-operative role for calbindin D-28k and NADPH-diaphorase in the functions of a subpopulation of vagal and glossopharyngeal sensory neurons.     

Selective activation of carotid nerve fibers by acetylcholine applied to the cat petrosal ganglion in vitro

The petrosal ganglion innervates carotid body chemoreceptors through the carotid (sinus) nerve. These primary sensory neurons are activated by transmitters released from receptor (glomus) cells, acetylcholine (ACh) having been proposed as one of the transmitters involved in this process. Since the perikarya of primary sensory neurons share several properties with peripheral sensory endings, we studied the electrical responses of the carotid nerve and glossopharyngeal branch to ACh locally applied to the cat petrosal ganglion superfused in vitro. Ganglionar applications of AChCl (1 μg−1 mg) generated bursts of action potentials conducted along the carotid nerve, while only a few spikes were exceptionally recorded from the glossopharyngeal branch in response to the largest doses. Carotid nerve responses to ACh were dose-dependent, the higher doses inducing transient desensitization. Application of nicotine to the petrosal ganglion also evoked dose-dependent excitatory responses in the carotid nerve. Responses to ACh were reversibly antagonized by adding hexamethonium to the superfusate, more intense and prolonged block of ACh responses being produced by mecamylamine. Ganglionar applications of γ-amino butyric acid and serotonin, in doses of up to 5 mg, did not induce firing of action potentials in any of the branches of the glossopharyngeal nerve. Our results indicate that petrosal ganglion neurons projecting through the carotid nerve are selectively activated by ACh acting on nicotinic ACh receptors located in the somata of these neurons. Thus, cholinosensitivity would be shared by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.     

Distribution of transient receptor potential channels in the rat carotid chemosensory pathway

Glomus cells in the carotid body respond to decreases in oxygen tension of the blood and transmit this sensory information in the carotid sinus nerve to the brain via neurons in the petrosal ganglion. G-protein-coupled membrane receptors linked to phospholipase C may play an important role in this response through the activation of the cation channels formed by the transient receptor potential (TRP) proteins. In the present study, expression of TRPC proteins in the rat carotid body and petrosal ganglion was examined using immunohistochemical techniques. TRPC3, TRPC4, TRPC5, TRPC6, and TRPC7 were present in neurons throughout the ganglion. TRPC1 was expressed in only 28% of petrosal neurons, and of this population, 45% were tyrosine hydroxylase (TH)-positive, accounting for essentially all the TH-expressing neurons in the ganglion. Because TH-positive neurons project to the carotid body, this result suggests that TRPC1 is selectively associated with the chemosensory pathway. Confocal images through the carotid body showed that TRPC1/3/4/5/6 proteins localize to the carotid sinus nerve fibers, some of which were immunoreactive to an anti-neurofilament (NF) antibody cocktail. TRPC1 and TRPC3 were present in both NF-positive and NF-negative fibers, whereas TPRC4, TRPC5, and TRPC6 expression was primarily localized to NF-negative fibers. Only TRPC1 and TRPC4 were localized in the afferent nerve terminals that encircle individual glomus cells. TRPC7 was not expressed in sensory fibers. All the TRPC proteins studied were present in type I glomus cells. Although their role as receptor-activated cation channels in the chemosensory pathway is yet to be established, the presence of TRPC channels in glomus cells and sensory nerves of the carotid body suggests a role in facilitating and/or sustaining the hypoxic response.     

Osteocalcin-immunoreactive neurons in the vagal and glossopharyngeal sensory ganglia of the rat

Immunohistochemistry for osteocalcin (OC) was performed on the rat vagal and glossopharyngeal sensory ganglia. OC-immunoreactive (IR) neurons were detected in the jugular (10%), petrosal (11%) and nodose ganglia (6%). The cell size analysis demonstrated that OC-IR neurons were predominantly small to medium-sized in the jugular ganglion (mean+/-S.D.=356.3+/-192.2 microm(2), range=86.5-831.5 microm(2)). On the other hand, such neurons were medium-sized to large in the petrosal (mean+/-S.D.=725.6+/-280.7 microm(2), range=124.7-1540.4 microm(2)) and nodose ganglia (mean+/-S.D.=857.5+/-330.2 microm(2), range=367.1-1608.0 microm(2)). In the circumvallate papilla, OC-IR nerve fibers were located in the vicinity of taste buds. Some taste bud cells were also immunoreactive for the calcium-binding protein (CaBP). In the carotid body, however, OC-IR nerve fibers could not be detected. Retrograde tracing with fluorogold revealed that OC-IR nerve fibers in the circumvallate papilla mainly originated from the petrosal ganglion. These findings may suggest that OC-IR petrosal neurons have chemoreceptive function in the tongue.     

Peptide 19 in the rat vagal and glossopharyngeal sensory ganglia

Peptide 19 (PEP 19) is a 7.6-kDa polypeptide which binds to calmodulin and inhibits calcium-calmodulin signaling. In this study, PEP 19-immunoreactivity (PEP 19-IR) was examined in the rat vagal and glossopharyngeal sensory ganglia. Twenty-nine percent, 59%, and 41% of sensory neurons contained PEP 19-IR in the jugular, petrosal, and nodose ganglia, respectively. These neurons were of various sizes (jugular, mean +/- SD = 635.8 +/- 392.6 microm2, range = 105.9-1695.9 microm2; petrosal, mean +/- SD = 370.9 +/- 228.5 microm2, range = 57.7-1662.7 microm2; nodose, mean +/- SD = 380.5 +/- 157 microm2, range = 87.5-950.4 microm2) and scattered throughout these ganglia. Double immunofluorescence method revealed that PEP 19-IR neurons which had parvalbumin-IR were rare in the ganglia (jugular, 4%; petrosal, 10%; nodose, 8%). PEP 19-IR neurons which contained calbindin D-28k were abundant in the petrosal (20%) and nodose (22%) ganglia but not in the jugular ganglion (8%). Retrograde tracing method indicated that many PEP 19-IR neurons projected to the circumvallate papilla and soft palate. In the soft palate, taste buds were innervated by PEP 19-IR nerve fibers. The present study suggests that PEP 19-IR neurons include chemoreceptors in the vagal and glossopharyngeal sensory ganglia.     

Retrograde neuronal labelling and double-staining immunohistochemistry of tachykinin- and calcitonin gene-related peptide-immunoreactive pathways in the carotid sinus nerve of the guinea pig

The origin of tachykinin- and calcitonin gene-related peptide-like immunoreactive (CGRP-LI) nerve fibres in the guinea pig carotid body and carotid sinus was determined by retrograde labelling of the carotid sinus nerve with Fluoro-gold and immunohistochemical double staining with fluorescein- and rhodamine-conjugated second antisera. Fluoro-gold-labelled perikarya with characteristic features of primary sensory neurones were numerous in the glossopharyngeal (petrosal) ganglion and occurred rarely in the closely attached superior vagal (jugular) ganglion. An efferent pathway from the brainstem could not be detected. Co-existence of tachykinin- and CGRP-LI was observed in 25-47% of labelled sensory neurones; less than 1% of Fluoro-gold-containing perikarya were exclusively stained by CGRP antiserum. Co-existence of tachykinin- and CGRP-LI was also demonstrated in nerve fibres of the carotid body and carotid sinus. Somatostatin-, cholecystokinin- and dynorphin-LI did not co-exist with tachykinin-LI in these fibres. Thus, tachykinin/CGRP-LI fibres in the carotid presso- and chemoreceptive areas exhibit a peptide pattern being generally characteristic for sensory fibres supplying great vessels in the guinea pig. In view of the present findings doubt is raised as to a primary involvement of these fibres in presso- or chemoreception, although a modulatory influence on these specific functions appears to be likely.     

Neurocalcin-immunoreactive neurons in the petrosal ganglion innervate the taste bud

The distribution and origin of neurocalcin-immunoreactive (NC-ir) nerve fibers in the taste bud and carotid body were examined by an immunofluorescence method. In the circumvallate papilla of the tongue, NC-ir nerve fibers made subepithelial nerve plexuses and occasionally penetrated the taste bud. However, the carotid body was devoid of ir nerve fibers. In the petrosal ganglion, 32% of neurons were immunoreactive for NC. Such neurons were mostly medium-sized to large, and scattered throughout the ganglion. In the superior cervical and intralingual ganglia, numerous ir varicose fibers surrounded postsynaptic neurons. However, NC-ir could not be detected in cell bodies of these neurons. The retrograde tracing method indicated that NC-ir petrosal neurons innervated taste buds in the circumvallate papilla. NC-ir neurons may have a gustatory function in the petrosal ganglion.     

Dopamine modulates carotid nerve responses induced by acetylcholine on the cat petrosal ganglion in vitro

We have recently reported that application of acetylcholine (ACh) or nicotine to the petrosal ganglion—the sensory ganglion of the glossopharyngeal nerve—elicits a burst of discharges in the carotid nerve branch, innervating the carotid body and sinus, but not in the glossopharyngeal branch, innervating the tongue and pharynx. Thus, the perikarya of sensory neurons for the carotid bifurcation exhibit selective cholinosensitivity. Since dopamine (DA) modulates carotid nerve chemosensory activity, we searched for the presence of DA sensitivity at the perikarya of these neurons in the cat petrosal ganglion superfused in vitro. Applications of DA in doses of up to 5 mg to the ganglion did not modify the rate of spontaneous discharges in the carotid nerve. However, if DA was applied 30 s before ACh injections, ACh-evoked reactions were modified: low doses of DA enhanced the subsequent responses to ACh, while high doses of DA depressed the responses to ACh. This depressant effect of DA on ACh responses was partially antagonized by adding spiroperone to the superfusate. Our results show that the response to ACh of petrosal ganglion neurons projecting through the carotid nerve is modulated by DA acting on D₂ receptors located in the somata of these neurons. Thus, dopaminergic modulation of cholinosensitivity could be shared also by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.     

Calretinin-immunoreactivity in vagal and glossopharyngeal sensory neurons of the rat: distribution and coexistence with putative transmitter agents.

Immunoreactivity for the calcium binding protein, calretinin (calretinin-ir), was demonstrated in cell bodies of vagal and glossopharyngeal sensory ganglia (jugular, petrosal, and nodose ganglia) and in associated nerve fibers. In the jugular and petrosal ganglia, many calretinin-ir neurons were also immunoreactive for calcitonin gene-related peptide and substance P. In the nodose ganglion, most of the calretinin-ir neurons lacked these peptides. None of the calretinin-ir neurons in these ganglia were also immunoreactive for tyrosine hydroxylase.     

The distribution of the cat''s carotid sinus nerve afferent and efferent cell bodies using the horseradish peroxidase technique

The location of both afferent and efferent carotid sinus nerve (CSN) cell bodies in the cat has been determined using the horseradish peroxidase (HRP) technique. Following a limited exposure of the central cut end of the CSN to HRP, labeled sensory ganglion cells were found in both the petrosal and superior ganglia of the IXth cranial nerve. An average of 387 in the former and 16 cells in the latter ganglion were labeled.

Retrogradely labeled neurons were found only within the ipsilateral medulla. These cells were both round and spindle shaped and had an average somal diameter of 19 μm. The number of these CSN efferent cell bodies ranged from 1 to a maximum of 20 in a given animal. They were found in both the nucleus parvocellularis and the retrofacial nucleus. In 8 cases axonal labeling was observed. Axons generally projected dorsomedially from the ventrolateral medulla.     

Delta-opioid receptor-immunoreactive neurons in the rat cranial sensory ganglia

Immunohistochemistry for delta-opioid receptor (DOR) was performed on the rat cranial sensory ganglia. The immunoreactivity was detected in 16%, 19% and 11% of neurons in the trigeminal, jugular and petrosal ganglia, respectively. The nodose ganglion was devoid of such neurons. DOR-immunoreactive (IR) neurons were mostly small to medium-sized (trigeminal, range = 62-851 microm(2), mean +/- SD = 359 +/- 175 microm(2); jugular, range = 120-854 microm(2), mean +/- SD = 409 +/- 196 microm(2); petrosal, range = 167-1146 microm(2), mean +/- SD = 423 +/- 233 microm(2)). Double immunofluorescence method revealed that all DOR-IR neurons were also immunoreactive for calcitonin gene-related peptide. The cutaneous and mucosal epithelia in the oro-facial region, tooth pulp, taste bud and carotid body were innervated by DOR-IR nerve fibers. In the brainstem, IR nerve terminals were located in the superficial medullary dorsal horn and dorsomedial part of the subnucleus oralis as well as the solitary tract nucleus. The present study suggests that DOR-IR neurons may be associated with nociceptive and/or chemoreceptive function in the cranial sensory ganglia.     

Application of shock waves to rat skin decreases calcitonin gene-related peptide immunoreactivity in dorsal root ganglion neurons

There have been several reports on the use of extracorporeal shock waves in the treatment of pseudarthrosis, calcifying tendinitis, and tendinopathies of the elbow. However, the pathomechanism of pain relief has not been clarified. To investigate the analgesic properties of shock wave application, we analyzed changes in calcitonin gene-related peptide (CGRP)-immunoreactive (ir) dorsal root ganglion (DRG) neurons. In the nontreated group, fluorogold-labeled dorsal root ganglion neurons innervating the most middle foot pad of hind paw were distributed in the L4 and L5 dorsal root ganglia. Of these neurons, 61% were CGRP-ir. However, in the shock wave-treated group, the percentage of FG-labeled CGRP-ir DRG neurons decreased to 18%. These data show that relief of clinical pain after shock wave application may result from reduced CGRP expression in DRG neurons.     

Partial coexistence of neuropeptide Y and calbindin D28k in the trigeminal ganglion following peripheral axotomy of the inferior alveolar nerve in the rat

Immunohistochemistry was applied to examine the correlation between neuropeptide Y (NPY) and the two calcium binding proteins (CaBPs) parvalbumin (PV) and calbindin D28k (CB) in the trigeminal ganglion following peripheral axotomy of the inferior alveolar nerve (IAN) in the rat. Five days following transection and application of FluoroGold (FG) to the cut end of the IAN, approximately 14.8% (80/539) and 18.6% (90/483) of FG-labeled IAN neurons in the trigeminal ganglion showed PV-like immunoreactivity (-LI) and CB-LI, respectively. The mean ± S.D. area of FG-labeled PV-like immunoreactive (-IR) cells (FG/PV-IR cells) and FG/CB-IR cells were 835.9 ± 303.1 μm² and 712.7 ± 246.0 μm², respectively. FG/PV-IR cells were significantly larger than FG/CB-IR cells. Fourteen days following peripheral axotomy of the IAN, NPY-LI appeared in the medium- to large-sized cells. Double immunostaining revealed that approximately 3.3% (52/1569) of NPY-IR cells in the axotomized trigeminal ganglion displayed PV-LI, while approximately 26.7% (371/1392) of NPY-IR cells displayed CB-LI. The mean ± S.D. cross-sectional areas of PV-IR and CB-IR trigeminal ganglion cells displaying NPY-LI were 819.5 ± 265.6 μm² and 766.5 ± 279.7 μm², respectively. There were no significant differences in the cross-sectional areas either between NPY/PV-IR cells and NPY/CB-IR cells, or between FG/PV-IR cells and NPY/PV-IR cells, or between FG/CB-IR cells and NPY/CB-IR cells. The present results indicate that injury-evoked medium- to large-sized NPY neurons were a different population from large-sized PV neurons, and NPY was partly co-localized with CB.