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Independent transmission of convergent visceral primary afferents in the solitary tract nucleus
Abstract
Cranial primary afferents from the viscera enter the brain at the solitary tract nucleus (NTS) where their information is integrated for homeostatic reflexes. The organization of sensory inputs is poorly understood despite its critical impact on overall reflex performance characteristics. Single afferents from the solitary tract (ST) branch within NTS and make multiple contacts onto individual neurons. Many neurons receive more than one ST input. To assess the potential interaction between converging afferents and proximal branching near to second order neurons, we probed near the recorded soma in horizontal slices from rats with focal electrodes and minimal shocks. Remote ST-shocks evoked monosynaptic excitatory postsynaptic currents (EPSCs) and nearby focal shocks also activated monosynaptic EPSCs. We tested the timing and order of stimulation to determine whether focal shocks influenced ST responses and vice versa in single neurons. Focal-evoked EPSCs response profiles closely resembled ST-EPSC characteristics. Mean synaptic jitters, failure rates, depression and phenotypic segregation by capsaicin responsiveness were indistinguishable between focal and ST evoked EPSCs. ST-EPSCs failed to affect focal-EPSCs within neurons indicating that release sites and synaptic terminals were functionally independent and isolated from crosstalk or neurotransmitter overflow. In only one instance, focal shocks intercepted and depleted the ST axon generating evoked EPSCs. Despite large numbers of functional contacts, multiple afferents do not appear to interact and ST axon branches may be limited to close to the soma. Thus, single or multiple primary afferents and their presynaptic active release sites act independently when they contact single second order NTS neurons.
... In no case did we detect evidence in the recruitment protocols for synaptic events that were of common ST origin in paired neighbouring neurons (i.e. synaptic events activated in both neurons with an identical threshold intensity consistent with activation of a single ST primary afferent diverging to two target neurons) (McDougall & Andresen, 2013). High-intensity ST shocks failed to evoke responses in four NTS-CeA neurons and two unlabelled neighbouring NTS cells. ...
... Our results suggest that the total number of CeA projection neurons is relatively restricted and the lack of dye-filled cells when injections missed their CeA target suggests a relatively delimited relationship between the caudal NTS and the amygdala. These NTS-CeA neurons received strongly convergent, ST-related inputs and clearly diverged from the more common pattern of neighbouring second-order NTS neurons, which tended to receive more limited and, not uncommonly, single ST afferent inputs (McDougall et al. 2009;Peters et al. 2011;McDougall & Andresen, 2013). Because unlabelled NTS second-order neurons were assayed simultaneously in the same slices as NTS-CeA neurons, any systematic ST damage from slicing probably does not account for these differences. ...
... In the physical layout within the NTS, myelinated and unmyelinated ST axons branch to spread their terminal fields broadly crossing anatomical subregions (Kubin et al. 1991;Kubin et al. 2006) and covering similar areas of NTS with no discernible spatial regionalization. Despite close proximity, adjacent second-order neurons received only one ST TRPV1 phenotype (McDougall & Andresen, 2013). TRPV1+ expression coincides with conduction velocities within the unmyelinated axon range and, correspondingly, TRPV1afferents conducted within the Aδ range ). ...
Key points:
Emotions are accompanied by concordant changes in visceral function, including cardiac output, respiration and digestion. One major forebrain integrator of emotional responses, the amygdala, is considered to rely on embedded visceral afferent information, although few details are known. In the present study, we retrogradely transported dye from the central nucleus of the amygdala (CeA) to identify CeA-projecting nucleus of the solitary tract (NTS) neurons for synaptic characterization and compared them with unlabelled, near-neighboor NTS neurons. Solitary tract (ST) afferents converged onto NTS-CeA second-order sensory neurons in greater numbers, as well as indirectly via polysynaptic pathways. Unexpectedly, all mono- and polysynaptic ST afferent pathways to NTS-CeA neurons were organized exclusively as either transient receptor potential cation channel subfamily V member 1 (TRPV1)-sensitive or TRPV1-resistant, regardless of whether intervening neurons were excitatory or inhibitory. This strict sorting provides viscerosensory signals to CeA about visceral conditions with respect to being either 'normal' via A-fibres or 'alarm' via TRPV1 expressing C-fibres and, accordingly, this pathway organization probably encodes interoceptive status.
Abstract:
Emotional state is impacted by changes in visceral function, including blood pressure, breathing and digestion. A main line of viscerosensory information processing occurs first in the nucleus of the solitary tract (NTS). In the present study conducted in rats, we examined the synaptic characteristics of visceral afferent pathways to the central nucleus of the amygdala (CeA) in brainstem slices by recording from retrogradely labelled NTS projection neurons. We simultaneously recorded neuron pairs: one dye positive (i.e. NTS-CeA) and a second unlabelled neighbour. Graded shocks to the solitary tract (ST) always (93%) triggered EPSCs at CeA projecting NTS neurons. Half of the NTS-CeA neurons received at least one primary afferent input (classed 'second order') indicating that viscerosensory information arrives at the CeA conveyed via a pathway involving as few as two synapses. The remaining NTS-CeA neurons received viscerosensory input only via polysynaptic pathways. By contrast, ∼3/4 of unlabelled neighbouring neurons were directly connected to ST. NTS-CeA neurons received greater numbers of ST-related inputs compared to unlabelled NTS neurons, indicating that highly convergent viscerosensory signals reach the CeA. Remarkably, despite multifibre convergence, all single NTS-CeA neurons received inputs derived from only unmyelinated afferents [transient receptor potential cation channel subfamily V member 1 (TRPV1) expressing C-fibres] or only non-TRPV1 ST afferent inputs, and never a combination of both. Such segregation means that visceral afferent information followed separate lines to reach the CeA. Their very different physiological activation profiles mean that these parallel visceral afferent pathways encode viscerosensory signals to the amygdala that may provide interoceptive assessments to impact on behaviours.
... That is, whilst second-order neurons may receive multiple A-fiber or multiple C-fiber inputs, it remains unclear whether a single second-order neuron receives both A-and C-fiber synaptic inputs. Earlier work in animal preparations would suggest that cross modal convergence is not only possible, but indeed widespread (283,308,357,358,400), whereas more recent studies employing brain stem slice recordings contradict these findings and support a model of modal segregation (298,299,363). Given that a wide variety of different sensory inputs can drive common respiratory and autonomic behaviors, it would seem that widespread convergence occurs at some juncture along these circuits, if not at the primary afferent synapse. ...
... Anatomical and functional studies suggest some degree of afferent convergence in the brain stem (220,283). Thus vagal afferent C-and A-fiber activation may synaptically drive common neurons in the nucleus of the solitary tract, although as discussed above it has also been debated as to whether any common second-order neurons are in direct convergent inputs (298). It is clear, however, that substance P increases the excitability of nucleus of the solitary tract neurons, thereby facilitating lung afferent transmission in guinea pigs and rabbits (283,324,325). ...
Vagal sensory neurons constitute the major afferent supply to the airways and lungs. Subsets of afferents are defined by their embryological origin, molecular profile, neurochemistry, functionality, and anatomical organization, and collectively these nerves are essential for the regulation of respiratory physiology and pulmonary defense through local responses and centrally mediated neural pathways. Mechanical and chemical activation of airway afferents depends on a myriad of ionic and receptor-mediated signaling, much of which has yet to be fully explored. Alterations in the sensitivity and neurochemical phenotype of vagal afferent nerves and/or the neural pathways that they innervate occur in a wide variety of pulmonary diseases, and as such, understanding the mechanisms of vagal sensory function and dysfunction may reveal novel therapeutic targets. In this comprehensive review we discuss historical and state-of-the-art concepts in airway sensory neurobiology and explore mechanisms underlying how vagal sensory pathways become dysfunctional in pathological conditions.
... It should be noted that the analysis in this paper primarily focusses on the'beat-to-beat'baroreflex, mediated primarily by A-fibers, characterized by myelinated axons with a lower activation threshold (approximately 60 mmHg) and near-constant activity throughout the cardiac cycle, encoding blood pressure levels through a frequency-modulated code of action potentials. A-fibers also feature a high conduction speed, relative to C-fibers, which are typically only activated during noxious or perilous stimuli, such as substantial increases in blood pressure, which may be characteristic of the Oxford method [17][18][19]. As a result, the results in this paper, and results from other non-invasive BRS studies, may not be directly comparable to those obtained using the invasive Oxford method for BRS estimation. ...
In the absence of a true gold standard for non-invasive baroreflex sensitivity estimation, it is difficult to quantify the accuracy of the variety of techniques used. A popular family of methods, usually entitled ‘sequence methods’ involves the extraction of (apparently) correlated sequences from blood pressure and RR-interval data and the subsequent fitting of a regression line to the data. This paper discusses the accuracy of sequence methods from a system identification perspective, using both data generated from a known mathematical model and spontaneous baroreflex data. It is shown that sequence methods can introduce significant bias in the baroreflex sensitivity estimate, even when great care is taken in sequence selection.
... Namely how specific or generalizable is the HFD-dysregulation to certain microcircuits. It has long been theorized that most second order NTS neurons represent a specific circuit since most NTS neurons receive inputs from only one or two afferent fibers 51,52 . However, whether a given NTS neuron communicates only with a single cardiac vagal motor neuron is currently unknown, and emerging evidence has only recently provided insight enabling construction of "projection models" of vagal motor neurons themselves 53,54 . ...
High fat diet (HFD) promotes cardiovascular disease and blunted cardiac vagal regulation. Temporal onset of loss of cardiac vagal control and its underlying mechanism are presently unclear. We tested our hypothesis that reduced central vagal regulation occurs early after HFD and contributes to poor cardiac regulation using cardiovascular testing paired with pharmacology in mice, molecular biology, and a novel bi-transgenic mouse line. Results show HFD, compared to normal fat diet (NFD), significantly blunted cardio/pulmonary chemoreflex bradycardic responses after 15 days, extending as far as tested (> 30 days). HFD produced resting tachycardia by day 3, reflected significant loss of parasympathetic tone. No differences in bradycardic responses to graded electrical stimulation of the distal cut end of the cervical vagus indicated diet-induced differences in vagal activity were centrally mediated. In nucleus ambiguus (NA), surface expression of δ-subunit containing type A gamma-aminobutyric acid receptors (GABAA(δ)R) increased at day 15 of HFD. Novel mice lacking δ-subunit expression in vagal motor neurons (ChAT-δnull) failed to exhibit blunted reflex bradycardia or resting tachycardia after two weeks of HFD. Thus, reduced parasympathetic output contributes to early HFD-induced HR dysregulation, likely through increased GABAA(δ)Rs. Results underscore need for research on mechanisms of early onset increases in GABAA(δ)R expression and parasympathetic dysfunction after HFD.
... Not all EPSCs evoked from single shocks to the T-2 nerve trunk had similar latency characteristics. Previous studies have suggested that the low jitter events represent direct, monosynaptic pathways to recorded CNS neurons with a cutoff of 200 μs (Doyle & Andresen, 2001;Fawley et al., 2021;McDougall & Andresen, 2013;McDougall et al., 2009). In SG neurons, T-2 activation also fell into low and high jitter categories using the 200 μs criterion. ...
The sympathetic nervous system vitally regulates autonomic functions, including cardiac activity. Postganglionic neurons of the sympathetic chain ganglia relay signals from the central nervous system to autonomic peripheral targets. Disrupting this flow of information often dysregulates organ function and leads to poor health outcomes. Despite the importance of these sympathetic neurons, fundamental aspects of the neurocircuitry within peripheral ganglia remain poorly understood. Conventionally, simple monosynaptic cholinergic pathways from preganglionic neurons are thought to activate postganglionic sympathetic neurons. However, early studies suggested more complex neurocircuits may be present within sympathetic ganglia. The present study recorded synaptic responses in sympathetic stellate ganglia neurons following electrical activation of the pre‐ and postganglionic nerve trunks and used genetic strategies to assess the presence of collateral projections between postganglionic neurons of the stellate ganglia. Orthograde activation of the preganglionic nerve trunk, T‐2, uncovered high jitter synaptic latencies consistent with polysynaptic connections. Pharmacological inhibition of nicotinic acetylcholine receptors with hexamethonium blocked all synaptic events. To confirm that high jitter, polysynaptic events were due to the presence of cholinergic collaterals from postganglionic neurons within the stellate ganglion, we knocked out choline acetyltransferase in adult noradrenergic neurons. This genetic knockout eliminated orthograde high jitter synaptic events and EPSCs evoked by retrograde activation. These findings suggest that cholinergic collateral projections arise from noradrenergic neurons within sympathetic ganglia. Identifying the contributions of collateral excitation to normal physiology and pathophysiology is an important area of future study and may offer novel therapeutic targets for the treatment of autonomic imbalance. image
Key points
Electrical stimulation of a preganglionic nerve trunk evoked fast synaptic transmission in stellate ganglion neurons with low and high jitter latencies.
Retrograde stimulation of a postganglionic nerve trunk evoked direct, all‐or‐none action currents and delayed nicotinic EPSCs indistinguishable from orthogradely‐evoked EPSCs in stellate neurons.
Nicotinic acetylcholine receptor blockade prevented all spontaneous and evoked synaptic activity.
Knockout of acetylcholine production in noradrenergic neurons eliminated all retrogradely‐evoked EPSCs but did not change retrograde action currents, indicating that noradrenergic neurons have cholinergic collaterals connecting neurons within the stellate ganglion.
... The second group included patients with the parasympathetic central nervous system disorders due to possible increased neurotropism, mainly of the vagus nerve and the olfactory bulb. This correlates with the data from other works, which put forward a different route of coronavirus infection migration, directly through the nerve trunks of the vagus nerve and the olfactory bulb [41][42][43][44]. ...
Поширення коронавірусної інфекції 2019 (COVID-19) спричинило пандемію, ефективних методів лікування й вакцин поки що немає. COVID-19 значною мірою впливає на багато органів і систем організму, включаючи серце, кишечник, нирки та мозок, незважаючи на те, що зазвичай у клінічній картині переважають пневмонія і легенева дисфункція. При вивченні статистики, структури захворюваності та механізмів порушення органів та систем у хворих на COVID-19 виявлено головну закономірність: який би орган або система не були пошкоджені, вегетативна нервова система обов’язково буде порушуватися, а це впливатиме на результат перебігу коронавірусної інфекції. У зв’язку з цим були виявлені різні біомаркери та концептуальні теорії, при аналізі та узагальненні яких назріла необхідність визначити стратегію профілактики та лікування вегетативних порушень.
... In second-order neurons, no time-dispersed or multiple eAP cases were encountered, indicating that any convergent afferent inputs were unable to doubly excite these second-order NTS neurons. This absence of A-and C-fibre convergence was consistent with both in vivo (Beaumont et al. 2017) and in vitro slice (McDougall & Andresen, 2013;Fawley et al. 2021) intracellular recordings of NTS neurons. In second-order NTS neurons, additional mechanisms that might prevent rapid re-excitation include a prolonged inhibition of spike generation by the common presence of feedback inhibitory synapses (Miles, 1986;Doyle & Andresen, 2001) or the presence of a strong A-type potassium current (Bailey et al. 2007). ...
Vagus nerve stimulation (VNS) treats patients with drug‐resistant epilepsy, depression and heart failure, but the mechanisms responsible are uncertain. The mild stimulus intensities used in chronic VNS suggest activation of myelinated primary visceral afferents projecting to the nucleus of the solitary tract (NTS). Here, we monitored the activity of second and higher order NTS neurons in response to peripheral vagal activation using therapeutic VNS criteria. A bipolar stimulating electrode activated the left cervical vagus nerve, and stereotaxically placed single tungsten electrodes recorded unit activity from the left caudomedial NTS of chloralose‐anaesthetized rats. High‐intensity single electrical stimuli established vagal afferent conduction velocity (myelinated A‐type or unmyelinated C‐type) as well as synaptic order (second vs. higher order using paired electrical stimuli) for inputs to single NTS neurons. Then, VNS treatment was applied. A mid‐collicular knife cut (KC) divided the brainstem from all supramedullary regions to determine their contribution to NTS activity. Our chief findings indicate that the KC reduced basal spontaneous activity of second‐order NTS neurons receiving myelinated vagal input by 85%. In these neurons, acute VNS increased activity similarly in Control and KC animals. Interestingly, the KC interrupted VNS activation of higher order NTS neurons and second‐order NTS neurons receiving unmyelinated vagal input, indicating that supramedullary descending projections to NTS are needed to amplify the peripheral neuronal signal from VNS. The present study begins to define the pathways activated during VNS and will help to better identify the central nervous system contributions to the therapeutic benefits of VNS therapy. image
Key points
Vagus nerve stimulation is routinely used in the clinic to treat epilepsy and depression, despite our uncertainty about how this treatment works.
For this study, the connections between the nucleus of the solitary tract (NTS) and the higher brain regions were severed to learn more about their contribution to activity of these neurons during stimulation.
Severing these brain connections reduced baseline activity as well as reducing stimulation‐induced activation for NTS neurons receiving myelinated vagal input.
Higher brain regions play a significant role in maintaining both normal activity in NTS and indirect mechanisms of enhancing NTS neuronal activity during vagus nerve stimulation.
... This higher order pathway structure is comparatively rare in the NTS (Bailey et al., 2006a;McDougall and Andresen, 2013). While half of the PVN-projecting neurons received at least one direct input to classify them as 2nd order neurons, most received at least one additional indirect input. ...
The nucleus of the solitary tract (NTS) receives viscerosensory information from the vagus nerve to regulate diverse homeostatic reflex functions. The NTS projects to a wide network of other brain regions, including the paraventricular nucleus of the hypothalamus (PVN). Here we examined the synaptic characteristics of primary afferent pathways to PVN-projecting NTS neurons in rat brainstem slices. Expression of the Transient Receptor Potential Vanilloid receptor (TRPV1+) distinguishes C-fiber afferents within the solitary tract (ST) from A-fibers (TRPV1-). We used resiniferatoxin (RTX), a TRPV1 agonist, to differentiate the two. The variability in the latency (jitter) of evoked excitatory postsynaptic currents (ST-EPSCs) distinguished monosynaptic from polysynaptic ST-EPSCs. Rhodamine injected into PVN was retrogradely transported to identify PVN-projecting NTS neurons within brainstem slices. Graded shocks to the ST elicited all-or-none EPSCs in rhodamine-positive NTS neurons with latencies that had either low jitter (<200 µs – monosynaptic), high jitter (>200 µs - polysynaptic inputs) or both. RTX blocked ST-evoked TRPV1+ EPSCs whether mono- or polysynaptic. Most PVN-projecting NTS neurons (17/21 neurons) had at least one input polysynaptically connected to the ST. Compared to unlabeled NTS neurons, PVN-projecting NTS neurons were more likely to receive indirect inputs and be higher order. Surprisingly, sEPSC rates for PVN-projecting neurons were double that of unlabeled NTS neurons. The ST synaptic responses for PVN-projecting NTS neurons were either all TRPV1+ or all TRPV1-, including neurons that received both direct and indirect inputs. Overall, PVN-projecting NTS neurons received direct and indirect vagal afferent information with strict segregation regarding TRPV1 expression.
... Furthermore, we recently identified two populations of paratrigeminal neurons, one expressing calbindin and a second expressing the NK1 receptor (Driessen et al. 2018). Thus, parallel non-convergent A and C fibre processing pathways may exist for afferent traffic through the paratrigeminal nucleus, similar to that described in the nucleus of the solitary tract (Bailey et al. 2002;McDougall & Andresen, 2013). ...
Key points
Airway projecting sensory neurons arising from the jugular vagal ganglia terminate centrally in the brainstem paratrigeminal nucleus, synapsing upon neurons expressing the neurokinin 1 receptor.
This study aimed to assess the involvement of paratrigeminal neurokinin 1 receptor neurons in the regulation of cough, breathing and airway defensive responses.
Lesioning neurokinin 1 receptor expressing paratrigeminal neurons significantly reduced cough evoked by inhaled bradykinin but not inhaled ATP or tracheal mechanical stimulation.
The reduction in bradykinin‐evoked cough was not accompanied by changes in baseline or evoked respiratory variables (e.g. frequency, volume or timing), animal avoidance behaviours or the laryngeal apnoea reflex.
These findings warrant further investigations into targeting the jugular ganglia and paratrigeminal nucleus as a therapy for treating cough in disease.
Abstract
Jugular vagal ganglia sensory neurons innervate the large airways and are thought to mediate cough and associated perceptions of airway irritations to a range of chemical irritants. The central terminals of jugular sensory neurons lie within the brainstem paratrigeminal nucleus, where postsynaptic neurons can be differentiated based on the absence or presence of the neurokinin 1 (NK1) receptor. Therefore, in the present study, we set out to test the hypothesis that NK1 receptor expressing paratrigeminal neurons play a role in cough evoked by inhaled chemical irritants. To test this, we performed selective neurotoxin lesions of NK1 receptor expressing neurons in the paratrigeminal nucleus in guinea‐pigs using substance P conjugated to saporin (SSP‐SAP). Sham lesion control or SSP‐SAP lesion guinea‐pigs received nebulised challenges, with the pan‐nociceptor stimulant bradykinin or the nodose ganglia specific stimulant adenosine 5′‐triphosphate (ATP), in conscious whole‐body plethysmography to study cough and associated behaviours. Laryngeal apnoea reflexes and cough evoked by mechanical stimulation of the trachea were additionally investigated in anaesthetised guinea‐pigs. SSP‐SAP significantly and selectively reduced the number of NK1 receptor expressing neurons in the paratrigeminal nucleus. This was associated with a significant reduction in bradykinin‐evoked cough, but not ATP‐evoked cough, mechanical cough or laryngeal apnoeic responses. These data provide further evidence for a role of jugular vagal pathways in cough, and additionally suggest an involvement of NK1 receptor expressing neurons in the paratrigeminal nucleus. Therefore, this neural pathway may provide novel therapeutic opportunities to treat conditions of chronic cough.
... Several lines of evidence suggest that the processing of afferent inputs within the paratrigeminal nucleus may be more complex than the simple notion that multiple afferent subtypes converge onto common post-synaptic paratrigeminal relay neurons. In this regard, the organisation of afferent inputs to the paratrigeminal nucleus may resemble the nonconvergent specificity of A-and C-fibre vagal afferent synapses that has previously been reported in the nucleus of the solitary tract (Bailey et al. 2002;McDougall and Andresen 2013). Indeed, whilst microinjection of a glutamate analogue predictably mimicked the effect of laryngeal stimulation by evoking a transient interruption of respiration (presumably secondary to the activation of post-synaptic targets), capsaicin microinjection into the paratrigeminal nucleus paradoxically enhanced respiratory drive. ...
Sensory neurons of the jugular vagal ganglia innervate the respiratory tract and project to the poorly studied medullary paratrigeminal nucleus. In the present study, we used neuroanatomical tracing, pharmacology and physiology in guinea pig to investigate the paratrigeminal neural circuits mediating jugular ganglia-evoked respiratory reflexes. Retrogradely traced laryngeal jugular ganglia neurons were largely (> 60%) unmyelinated and expressed the neuropeptide substance P and calcitonin gene-related peptide, although a population (~ 30%) of larger diameter myelinated jugular neurons was defined by the expression of vGlut1. Within the brainstem, vagal afferent terminals were confined to the caudal two-thirds of the paratrigeminal nucleus. Electrical stimulation of the laryngeal mucosa evoked a vagally mediated respiratory slowing that was mimicked by laryngeal capsaicin application. These laryngeal reflexes were modestly reduced by neuropeptide receptor antagonist microinjections into the paratrigeminal nucleus, but abolished by ionotropic glutamate receptor antagonists. d,l-Homocysteic acid microinjections into the paratrigeminal nucleus mimicked the laryngeal-evoked respiratory slowing, whereas capsaicin microinjections evoked a persistent tachypnoea that was insensitive to glutamatergic inhibition but abolished by neuropeptide receptor antagonists. Extensive projections from paratrigeminal neurons were anterogradely traced throughout the pontomedullary respiratory column. Dual retrograde tracing from pontine and ventrolateral medullary termination sites, as well as immunohistochemical staining for calbindin and neurokinin 1 receptors, supported the existence of different subpopulations of paratrigeminal neurons. Collectively, these data provide anatomical and functional evidence for at least two types of post-synaptic paratrigeminal neurons involved in respiratory reflexes, highlighting an unrecognised complexity in sensory processing in this region of the brainstem.
... Neurons in the NTS and adjacent medial medulla have heterogeneous phenotypes and diverse responses to carotid body stimulation (2, 4, 36, 116,120,157,194,265). These "chemoresponsive" neurons operate through multiple circuit pathways to regulate the depth and frequency of breathing and, concurrently, cardiac output and vascular tone (FIGURE 1) (91, 177). ...
Advances in our understanding of brain mechanisms for the hypoxic ventilatory response, coordinated changes in blood pressure, and the long-term consequences of chronic intermittent hypoxia as in sleep apnea, such as hypertension and heart failure, are giving impetus to the search for therapies to "erase" dysfunctional memories distributed in the carotid bodies and central nervous system. We review current network models, open questions, sex differences, and implications for translational research.
... The innervation of the diaphragm muscle may be directly responsible for the emotional state of the patient through the phrenic nerve, not only due to the interoceptive mechanism. The afferent stimulation to the NTS by the phrenic nerve could affect the emotional response, because NTS handles the visceral afferents and has a close relationship with the nerve [28]. The phrenic nerve forms subdiaphragmatic ganglia and is connected with the adrenal gland; anyway, further data even on these connections are currently needed. ...
... The NTS contains three distinct glutamate signals at TRPV1+ ST afferents: evoked, spontaneous, and asynchronous release [9], and these forms of transmission stand on a solid foundation of work [4,14,16,17,19,22,[32][33][34]. Each type of release depends on increases in calcium but from different sources: spontaneous release requires calcium influx via TRPV1 while evoked and asynchronous release depends on calcium increases via VGCCs [4]. ...
The recycling of vesicle membrane fused during exocytosis is essential to maintaining neurotransmission. The GTPase dynamin is involved in pinching off membrane to complete endocytosis and can be inhibited by dynasore resulting in activity-dependent depletion of release-competent synaptic vesicles. In rat brainstem slices, we examined the effects of dynasore on three different modes of glutamate release–spontaneous, evoked, and asynchronous release–at solitary tract (ST) inputs to neurons in the nucleus of the solitary tract (NTS). Intermittent bursts of stimuli to the ST interspersed with pauses in stimulation allowed examination of these three modes in each neuron continuously. Application of 100 μM dynasore rapidly increased the spontaneous EPSC (sEPSC) frequency which was followed by inhibition of both ST-evoked EPSCs (ST-EPSC) as well as asynchronous EPSCs. The onset of ST-EPSC failures was not accompanied by amplitude reduction–a pattern more consistent with conduction block than reduced probability of vesicle release. Neither result suggested that dynasore interrupted endocytosis. The dynasore response profile resembled intense presynaptic TRPV1 activation. The TRPV1 antagonist capsazepine failed to prevent dynasore increases in sEPSC frequency but did prevent the block of the ST-EPSC. In contrast, the TRPV1 antagonist JNJ 17203212 prevented both actions of dynasore in neurons with TRPV1-expressing ST inputs. In a neuron lacking TRPV1-expressing ST inputs, however, dynasore promptly increased sEPSC rate followed by block of ST-evoked EPSCs. Together our results suggest that dynasore actions on ST-NTS transmission are TRPV1-independent and changes in glutamatergic transmission are not consistent with changes in vesicle recycling and endocytosis.
... 63 For further characteristic features of these vagal sensory neurons including suggestion of phenotyping segregation by capsaicin responsiveness are summarized in this and in another paper of this group. 64 Thus, several recent reports provide evidence that TRPV1expressing capsaicin-sensitive visceral primary afferent neurons, their thoracic or abdominal receptors and central terminals could serve as thermoreceptors within the physiological range of thermoregulation. More efforts are needed, however, to clarify their role how they regulate various effectors and behavioral responses in thermal homeostasis. ...
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... | Neuroendocrine Science of action potential driven synchronous EPSCs enhanced the TRPV1-operated vesicle release for a period of seconds and produced asynchronous release sufficient to trigger additional action potentials in the postsynaptic neuron (Peters et al., 2010Peters et al., , 2011 McDougall and Andresen, 2013 ) in basal conditions that are incremented even higher in an asynchronous mode of release. The physiological meaning of the TRPV1-operated pool of vesicles is only beginning to be appreciated. ...
The brainstem nucleus of the solitary tract (NTS) holds the first central neurons in major homeostatic reflex pathways. These homeostatic reflexes regulate and coordinate multiple organ systems from gastrointestinal to cardiopulmonary functions. The core of many of these pathways arise from cranial visceral afferent neurons that enter the brain as the solitary tract (ST) with more than two-thirds arising from the gastrointestinal system. About one quarter of ST afferents have myelinated axons but the majority are classed as unmyelinated C-fibers. All ST afferents release the fast neurotransmitter glutamate with remarkably similar, high-probability release characteristics. Second order NTS neurons receive surprisingly limited primary afferent information with one or two individual inputs converging on single second order NTS neurons. A- and C-fiber afferents never mix at NTS second order neurons. Many transmitters modify the basic glutamatergic excitatory postsynaptic current often by reducing glutamate release or interrupting terminal depolarization. Thus, a distinguishing feature of ST transmission is presynaptic expression of G-protein coupled receptors for peptides common to peripheral or forebrain (e.g., hypothalamus) neuron sources. Presynaptic receptors for angiotensin (AT1), vasopressin (V1a), oxytocin, opioid (MOR), ghrelin (GHSR1), and cholecystokinin differentially control glutamate release on particular subsets of neurons with most other ST afferents unaffected. Lastly, lipid-like signals are transduced by two key ST presynaptic receptors, the transient receptor potential vanilloid type 1 and the cannabinoid receptor that oppositely control glutamate release. Increasing evidence suggests that peripheral nervous signaling mechanisms are repurposed at central terminals to control excitation and are major sites of signal integration of peripheral and central inputs particularly from the hypothalamus.
The hormone leptin reduces food intake through actions in the peripheral and central nervous system, including in the hindbrain nucleus of the solitary tract (NTS). The NTS receives viscerosensory information via vagal afferents, including information from the gastrointestinal tract, which is then relayed to other CNS sites critical for control of food intake. Leptin receptors (lepRs) are expressed by a subpopulation of NTS neurons, and knockdown of these receptors increases both food intake and body weight. Recently, we demonstrated that leptin increases vagal activation of lepR-expressing neurons via increased NMDAR currents, thereby potentiating vagally-evoked firing. Furthermore, chemogenetic activation of these neurons was recently shown to inhibit food intake. However, the vagal inputs these neurons receive had not been characterized. Here we performed whole-cell recordings in brain slices taken from lepRCre X floxedTdTomato mice and found that lepR neurons of the NTS are directly activated by monosynaptic inputs from C-type afferents sensitive to the TRPV1 agonist capsaicin. CCK administered onto NTS slices stimulated spontaneous glutamate release onto lepR neurons and induced action potential firing; an effect mediated by CCKR 1 . Interestingly, NMDAR activation contributed to the current carried by spontaneous EPSCs and enhanced CCK-induced firing. Peripheral CCK also increased c-fos expression in these neurons, suggesting they are activated by CCK-sensitive vagal afferents in vivo. Our results indicate that the majority of NTS lepR neurons receive direct inputs from CCK-sensitive C vagal type afferents, with both peripheral and central CCK capable of activating these neurons and NMDARs able to potentiate these effects.
Airway afferents monitor the local chemical and physical micro-environments in the airway wall and lungs and send this information centrally to regulate neural circuits involved in setting autonomic tone, evoking reflex and volitional respiratory motor outflows, encoding perceivable sensations and contributing to higher order cognitive processing. In this mini-review we present a current overview of the central wiring of airway afferent circuits in the brainstem and brain, highlighting recent discoveries that augment our understanding of airway sensory processing. We additionally explore how advances in describing the molecular diversity of airway afferents may influence future research efforts aimed at defining central mesoscale connectivity of airway afferent pathways. A refined understanding of how functionally distinct airway afferent pathways are organized in the brain will provide deeper insight into the physiology of airway afferent-evoked responses and may foster opportunities for targeted modulation of specific pathways involved in disease.
Muscle sympathetic single units can respond differentially to stress but whether these responses are linked to the degree of sympathoexcitation is unclear. Fifty-three muscle sympathetic single units (microneurography) were recorded in 17 participants (8 women; 24±3 yr). Five 40-second bouts of 10% static handgrip were performed during a 10 min forearm ischemia to progressively increase metabolite accumulation. Each static handgrip was separated by a 75 sec ischemic rest (post-exercise circulatory occlusion [PECO]) to assess the isolated action of the muscle metaboreflex. During each set of PECO, individual single units were classified as activated, non-responsive, or inhibited if the spike frequency was above, within, or below the baseline variability, respectively. From sets 1-5 of PECO, the proportion of single units with activated (34, 45, 68, 87, 89%), non-responsive (43, 44, 23, 7, 9%), or inhibited (23, 11, 9, 6, 2%) responses changed (P<0.001) as total muscle sympathoexcitation increased. 51/53 (96%) single units were activated in at least one set of PECO, 16 (31%) initially inhibited prior to activation. This response pattern delayed the activation onset compared to non-inhibited units (set 3±1 vs. 2±1, P<0.001). Once activated, the spike frequency rate-of-rise was similar (8.5±6.5 vs. 7.1±6.0 spikes/min/set, P=0.48). Muscle sympathetic single-unit firing demonstrated differential control during muscle metaboreflex activation. Single units that were initially inhibited during progressive metaboreflex activation were capable of being activated in later sets. These findings reveal that single-unit activity is influenced by convergent neural inputs (i.e. both inhibitory and excitatory), which yield heterogenous single-unit activation thresholds.
Cough is an important protective mechanism for clearing the airways but becomes a troublesome, and often difficult to treat, symptom in respiratory disease. Although cough can be produced as a reflex in response to the presence of irritants within the airways, emerging research demonstrates an unappreciated complexity in the peripheral and central neural systems that regulate cough. This complexity includes multiple primary sensory neurons that can induce or facilitate reflex coughing, different ascending central circuits in the brain that contribute to cough sensory discrimination and the perception of the urge-to-cough, and several descending brain systems for inducing, facilitating and inhibiting cough responses. Consequently, the mechanisms responsible for cough becoming dysregulated in disease are not likely homogeneous across all patients with chronic cough. The available data suggests that changes in primary sensory neuron excitability, altered central nervous system integration of sensory inputs and changes in descending control mechanisms may each contribute to the development of cough hypersensitivity.
Homeostatic regulation of visceral organ function requires integrated processing of neural and neurohormonal sensory signals. The nucleus of the solitary tract (NTS) is the primary sensory nucleus for cranial visceral sensory afferents. Angiotensin II (Ang II) is known to modulate peripheral visceral reflexes, in part, by activating Ang II type 1A receptors (AT1AR) in the NTS. AT1AR-expressing NTS neurons occur throughout the NTS with a defined sub-nuclear distribution and most of these neurons are depolarized by Ang II. In this study we determined whether AT1AR-expressing NTS neurons receive direct visceral sensory input, and whether this input is modulated by Ang II. Using AT1AR-GFP mice to make targeted whole cell recordings from AT1AR-expressing NTS neurons, we demonstrate that two thirds (37 of 56) of AT1AR-expressing neurons receive direct excitatory, visceral sensory input. In half of the neurons tested (4 of 8) the excitatory visceral sensory input was significantly reduced by application of the transient receptor potential vallinoid type 1 receptor agonist, capsaicin, indicating AT1AR-expressing neurons can receive either C or A-fibre mediated input. Application of Ang II to a subset of second order AT1AR-expressing neurons did not affect spontaneous, evoked or asynchronous glutamate release from visceral sensory afferents. Thus it is unlikely that AT1AR-expressing viscerosensory neurons terminate on AT1AR-expressing NTS neurons. Our data suggest that Ang II is likely to modulate multiple visceral sensory modalities by altering the excitability of second order AT1AR-expressing NTS neurons.
Unlabelled:
Most craniosensory afferents have unmyelinated axons expressing TRP Vanilloid 1 (TRPV1) receptors in synaptic terminals at the solitary tract nucleus (NTS). Neurotransmission from these synapses is characterized by substantial asynchronous EPSCs following action potential-synched EPSCs and high spontaneous rates that are thermally sensitive. The present studies blocked voltage-activated calcium channels (CaV) using the nonselective CaV blocker Cd(2+) or the specific N-type blocker ω-conotoxin GVIA to examine the calcium dependence of the synchronous, asynchronous, spontaneous, and thermally gated modes of release. In rat brainstem slices containing caudal NTS, shocks to the solitary tract (ST) triggered synchronous ST-EPSCs and trailing asynchronous EPSCs. Cd(2+) or GVIA efficiently reduced both synchronous and asynchronous EPSCs without altering spontaneous or thermal-evoked transmission. Activation of TRPV1 with either the selective agonist resiniferatoxin (150 pm) or temperature augmented basal sEPSC rates but failed to alter the synchronous or asynchronous modes of release. These data indicate that calcium sourced through TRPV1 has no access to the synchronous or asynchronous release mechanism(s) and conversely that CaV-sourced calcium does not interact with the thermally evoked mode of release. Buffering intracellular calcium with EGTA-AM or BAPTA-AM reduced asynchronous EPSC rates earlier and to a greater extent than synchronous ST-EPSC amplitudes without altering sEPSCs or thermal sensitivity. Buffering therefore distinguishes asynchronous vesicles as possessing a highly sensitive calcium sensor located perhaps more distant from CaV than synchronous vesicles or thermally evoked vesicles from TRPV1. Together, our findings suggest separate mechanisms of release for spontaneous, asynchronous and synchronous vesicles that likely reside in unique, spatially separated vesicle domains.
Significance statement:
Most craniosensory fibers release glutamate using calcium entry from two sources: CaVs and TRPV1. We demonstrate that calcium segregation distinguishes three vesicle release mechanisms. Most surprisingly, asynchronous release is associated with CaV and not TRPV1 calcium entry. This reveals that asynchronous release is an additional and separate phenotypic marker of unmyelinated afferents rather than operated by TRPV1. The functional independence of the two calcium sources expands the regulatory repertoire of transmission and imbues these inputs with additional modulation targets for synaptic release not present at conventional CaV synapses. Peptides and lipid mediators may target one or both of these calcium sources at afferent terminals within the solitary tract nucleus to independently modify release from distinct, functionally segregated vesicle pools.
Action potentials trigger synaptic terminals to synchronously release vesicles, but some vesicles release spontaneously. G-protein-coupled receptors (GPCRs) can modulate both of these processes. At cranial primary afferent terminals, the GPCR cannabinoid 1 (CB1) is often coexpressed with transient receptor potential vanilloid 1 (TRPV1), a nonselective cation channel present on most afferents. Here we tested whether CB1 activation modulates synchronous, action potential-evoked (eEPSCs) and/or spontaneous (sEPSCs) EPSCs at solitary tract nucleus neurons. In rat horizontal brainstem slices, activation of solitary tract (ST) primary afferents generated ST-eEPSCs that were rapidly and reversibly inhibited from most afferents by activation of CB1 with arachidonyl-2'-chloroethylamide (ACEA) or WIN 55,212-2 [R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone monomethanesulfonate]. The CB1 antagonist/inverse agonist AM251 [N-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] blocked these responses. Despite profound depression of ST-eEPSCs during CB1 activation, sEPSCs in these same neurons were unaltered. Changes in temperature changed sEPSC frequency only from TRPV1(+) afferents (i.e., thermal sEPSC responses only occurred in TRPV1(+) afferents). CB1 activation failed to alter these thermal sEPSC responses. However, the endogenous arachidonate metabolite N-arachidonyldopamine (NADA) promiscuously activated both CB1 and TRPV1 receptors. NADA inhibited ST-eEPSCs while simultaneously increasing sEPSC frequency, and thermally triggered sEPSC increases in neurons with TRPV1(+) afferents. We found no evidence for CB1/TRPV1 interactions suggesting independent regulation of two separate vesicle pools. Together, these data demonstrate that action potential-evoked synchronous glutamate release is modulated separately from TRPV1-mediated glutamate release despite coexistence in the same central terminations. This two-pool arrangement allows independent and opposite modulation of glutamate release by single lipid metabolites.
Visual information must be relayed through the lateral geniculate nucleus before it reaches the visual cortex. However, not all spikes created in the retina lead to postsynaptic spikes and properties of the retinogeniculate synapse contribute to this filtering. To understand the mechanisms underlying this filtering process, we conducted electrophysiology to assess the properties of signal transmission in the Long-Evans rat. We also performed SDS-digested freeze-fracture replica labeling to quantify the receptor and transporter distribution, as well as EM reconstruction to describe the 3D structure. To analyze the impact of transmitter diffusion on the activity of the receptors, simulations were integrated. We identified that a large contributor to the filtering is the marked paired-pulse depression at this synapse, which was intensified by the morphological characteristics of the contacts. The broad presynaptic and postsynaptic contact area restricts transmitter diffusion two dimensionally. Additionally, the presence of multiple closely arranged release sites invites intersynaptic spillover, which causes desensitization of AMPA receptors. The presence of AMPA receptors that slowly recover from desensitization along with the high presynaptic release probability and multivesicular release at each synapse also contribute to the depression. These features contrast with many other synapses where spatiotemporal spread of transmitter is limited by rapid transmitter clearance allowing synapses to operate more independently. We propose that the micrometer-order structure can ultimately affect the visual information processing.
TRPV1 receptors are expressed on most but not all central terminals of cranial visceral afferents in the caudal solitary tract nucleus (NTS). TRPV1 is associated with unmyelinated C-fiber afferents. Both TRPV1+ and TRPV1- afferents enter NTS but their precise organization remains poorly understood. In horizontal brainstem slices, we activated solitary tract (ST) afferents and recorded ST-evoked glutamatergic excitatory synaptic currents (ST-EPSCs) under whole cell voltage clamp conditions from neurons of the medial subnucleus. Electrical shocks to the ST produced fixed latency EPSCs (jitter<200 µs) that identified direct ST afferent innervation. Graded increases in shock intensity often recruited more than one ST afferent and ST-EPSCs had consistent threshold intensity, latency to onset, and unique EPSC waveforms that characterized each unitary ST afferent contact. The TRPV1 agonist capsaicin (100 nM) blocked the evoked TRPV1+ ST-EPSCs and defined them as either TRPV1+ or TRPV1- inputs. No partial responses to capsaicin were observed so that in NTS neurons that received one or multiple (2-5) direct ST afferent inputs--all were either blocked by capsaicin or were unaltered. Since TRPV1 mediates asynchronous release following TRPV1+ ST-evoked EPSCs, we likewise found that recruiting more than one ST afferent further augmented the asynchronous response and was eliminated by capsaicin. Thus, TRPV1+ and TRPV1- afferents are completely segregated to separate NTS neurons. As a result, the TRPV1 receptor augments glutamate release only within unmyelinated afferent pathways in caudal medial NTS and our work indicates a complete separation of C-type from A-type afferent information at these first central neurons.
The heat and capsaicin receptor, TRPV1, is required for the detection of painful heat by primary afferent pain fibers (nociceptors), but the extent to which functional TRPV1 channels are expressed in the CNS is debated. Because previous evidence is based primarily on indirect physiological responses to capsaicin, here we genetically modified the Trpv1 locus to reveal, with excellent sensitivity and specificity, the distribution of TRPV1 in all neuronal and non-neuronal tissues. In contrast to reports of widespread and robust expression in the CNS, we find that neuronal TRPV1 is primarily restricted to nociceptors in primary sensory ganglia, with minimal expression in a few discrete brain regions, most notably in a contiguous band of cells within and adjacent to the caudal hypothalamus. We confirm hypothalamic expression in the mouse using several complementary approaches, including in situ hybridization, calcium imaging, and electrophysiological recordings. Additional in situ hybridization experiments in rat, monkey, and human brain demonstrate that the restricted expression of TRPV1 in the CNS is conserved across species. Outside of the CNS, we find TRPV1 expression in a subset of arteriolar smooth muscle cells within thermoregulatory tissues. Here, capsaicin increases calcium uptake and induces vasoconstriction, an effect that likely counteracts the vasodilation produced by activation of neuronal TRPV1.
Brainstem A2/C2 catecholamine (CA) neurons in the solitary tract nucleus (NTS) are thought to play an important role in the control of food intake and other homeostatic functions. We have previously demonstrated that these neurons, which send extensive projections to brain regions involved in the regulation of appetite, are strongly and directly activated by solitary tract (ST) visceral afferents. Ghrelin, a potent orexigenic peptide released from the stomach, is proposed to act in part through modulating NTS CA neurons but the underlying cellular mechanisms are unknown. Here, we identified CA neurons using transgenic mice that express enhanced green fluorescent protein driven by the tyrosine hydroxylase promoter (TH-EGFP). We then determined how ghrelin modulates TH-EGFP neurons using patch-clamp techniques in a horizontal brain slice preparation. Ghrelin inhibited the frequency of spontaneous glutamate inputs (spontaneous EPSCs) onto TH-EGFP neurons, including cholecystokinin-sensitive neurons, an effect blocked by the GHSR1 antagonist, d-Lys-3-GHRP-6. This resulted in a decrease in the basal firing rate of NTS TH-EGFP neurons, an effect blocked by the glutamate antagonist NBQX. Ghrelin also dose-dependently inhibited the amplitude of ST afferent evoked EPSCs (ST-EPSCs) in TH-EGFP NTS neurons, decreasing the success rate for ST-evoked action potentials. In addition, ghrelin decreased the frequency of mini-EPSCs suggesting its actions are presynaptic to reduce glutamate release. Last, inhibition by ghrelin of the ST-EPSCs was significantly increased by an 18 h fast. These results demonstrate a potential mechanism by which ghrelin inhibits NTS TH neurons through a pathway whose responsiveness is increased during fasting.
Primary afferent axons within the solitary tract (ST) relay homeostatic information via glutamatergic synapses directly to second-order neurons within the nucleus of the solitary tract (NTS). These primary afferents arise from multiple organ systems and relay multiple sensory modalities. How this compact network organizes the flow of primary afferent information will shape central homeostatic control. To assess afferent convergence and divergence, we recorded ST-evoked synaptic responses in pairs of medial NTS neurons in horizontal brainstem slices. ST shocks activated EPSCs along monosynaptic or polysynaptic pathways. Gradations in shock intensity discriminated multiple inputs and stimulus recruitment profiles indicated that each EPSC was unitary. In 24 pairs, 75% were second-order neurons with 64% receiving one direct ST input with the remainder receiving additional convergent ST afferent inputs (22% two; 14% three monosynaptic ST-EPSCs). Some (34%) second-order neurons received polysynaptic EPSCs. Neurons receiving only higher-order inputs were uncommon (13%). Most ST-EPSCs were completely independent, but 4 EPSCs of a total of 81 had equal thresholds, highly correlated latencies, and synchronized synaptic failures consistent with divergence from a single source ST axon or from a common interneuron producing a pair of polysynaptic EPSCs. We conclude that ST afferent inputs are remarkably independent with little evidence of substantial shared information. Individual cells receive highly focused information from the viscera. Thus, afferent excitation of second-order NTS neurons is generally dominated by single visceral afferents and therefore focused on a single afferent modality and/or organ region.
At a number of synapses, long-term potentiation (LTP) can be expressed by an increase in presynaptic strength, but it is unknown whether presynaptic LTP is expressed solely through an increase in the probability that a single vesicle is released or whether it can increase multivesicular release (MVR). Here, we show that presynaptic LTP decreases inhibition of AMPA receptor EPSCs by a low-affinity antagonist at parallel fiber-molecular layer interneuron (PF-MLI) synapses. This indicates that LTP induction results in larger glutamate concentration transients in the synaptic cleft, a result indicative of MVR, and suggests that MVR can be modified by long-term plasticity. A similar decrease in inhibition was observed when release probability (PR) was increased by forskolin, elevated extracellular Ca2+, and paired-pulse facilitation. Furthermore, we show that MVR may occur under baseline physiological conditions, as inhibition increased when P(R) was lowered by reducing extracellular Ca2+ or by activating presynaptic adenosine receptors. These results suggest that at PF-MLI synapses, MVR occurs under control conditions and is increased when PR is elevated by both short- and long-term plasticity mechanisms.
Cranial visceral afferents travel via the solitary tract (ST) to contact neurons within the ST nucleus (NTS) and activate homeostatic reflexes. Hypothalamic projections from the paraventricular nucleus (PVN) release oxytocin (OT) to modulate visceral afferent communication with NTS neurons. However, the cellular mechanisms through which OT acts are poorly understood. Here, we electrophysiologically identified second-order NTS neurons in horizontal brainstem slices by their low-jitter, ST-evoked glutamatergic EPSCs. OT increased the frequency of miniature EPSCs in half of the NTS second-order neurons (13/24) but did not alter event kinetics or amplitudes. These actions were blocked by a selective OT receptor antagonist. OT increased the amplitude of ST-evoked EPSCs with no effect on event kinetics. Variance-mean analysis of ST-evoked EPSCs indicated OT selectively increased the release probability of glutamate from the ST afferent terminals. In OT-sensitive neurons, OT evoked an inward holding current and increased input resistance. The OT-sensitive current reversed at the K(+) equilibrium potential. In in vivo studies, NTS neurons excited by vagal cardiopulmonary afferents were juxtacellularly labeled with Neurobiotin and sections were stained to show filled neurons and OT-immunoreactive axons. Half of these physiologically characterized neurons (5/10) showed close appositions by OT fibers consistent with synaptic contacts. Electron microscopy of medial NTS found immunoreactive OT within synaptic boutons. Together, these findings suggest that OT released from PVN axons acts on a subset of second-order neurons within medial NTS to enhance visceral afferent transmission via presynaptic and postsynaptic mechanisms.
Transmission at individual synaptic contacts on CA1 hippocampal pyramidal neurons has been found to be very unreliable, with greater than half of the arriving presynaptic nerve impulses failing to evoke a postsynaptic response. This conclusion has been reached using the method of minimal stimulation of Schaffer collaterals and whole cell recording in hippocampal slices; with minimal stimulation only one or a few synapses are activated on the target neuron and the behavior of individual synapses can be examined. Four sources for the unreliability of synaptic transmission have been investigated: (i) the fluctuation of axon thresholds at the site of stimulation causing the failure to generate a nerve impulse in the appropriate Schaffer collaterals, (ii) the failure of nerve impulses generated at the site of stimulation to arrive at the synapse because of conduction failures at axon branch points, (iii) an artifactual synaptic unreliability due to performing experiments in vitro at temperatures well below the normal mammalian body temperature, and (iv) transmission failures due to probabilistic release mechanisms at synapses with a very low capacity to release transmitter. We eliminate the first three causes as significant contributions and conclude that probabilistic release mechanisms at low capacity synapses are the main cause of unreliability of synaptic transmission.
The frequency of baroreceptor volleys to the central nervous system can influence the fidelity of baroreceptor signal transmission and thus may affect baroreflex regulation of blood pressure. We examined 1) the extent to which frequency-dependent depression of aortic baroreceptor signals was initiated at the first central synapse between primary baroreceptor fibers and second-order nucleus tractus solitarii (NTS) neurons; 2) whether the pattern of baroreceptor input influenced the depression; and 3) the potential relevance to baroreflex sympathoinhibition. In urethan-anesthetized rats, NTS action potential responses of neurons classified as second or higher order and averaged lumbar sympathetic nerve activity responses were simultaneously measured during 100 aortic depressor nerve stimuli delivered in constant or phasic patterns (0.8-48 Hz). Frequency-dependent depression was initiated at second-order neurons, with NTS responses decreasing to a 72% response rate at 48 Hz; the depression was greater at higher-order neurons; responses decreased to a 30% response rate. The depression was slightly but significantly greater with phasic inputs. Curve fitting suggested that synaptic depression may limit baroreflex sympathoinhibition. Thus frequency limits on baroreceptor inputs at NTS synapses may affect baroreflex function.
The timing of events within the nervous system is a critical feature of signal processing and integration. In neurotransmission, the synaptic latency, the time between stimulus delivery and appearance of the synaptic event, is generally thought to be directly related to the complexity of that pathway. In horizontal brain stem slices, we examined synaptic latency and its shock-to-shock variability (synaptic jitter) in medial nucleus tractus solitarius (NTS) neurons in response to solitary tract (ST) electrical activation. Using a visualized patch recording approach, we activated ST 1-3 mm from the recorded neuron with short trains (50-200 Hz) and measured synaptic currents under voltage clamp. Latencies ranged from 1.5 to 8.6 ms, and jitter values (SD of intraneuronal latency) ranged from 26 to 764 micros (n = 49). Surprisingly, frequency of synaptic failure was not correlated with either latency or jitter (P > 0.147; n = 49). Despite conventional expectations, no clear divisions in latency were found from the earliest arriving excitatory postsynaptic currents (EPSCs) to late pharmacologically polysynaptic responses. Shortest latency EPSCs (<3 ms) were mediated by non-N-methyl-D-aspartate (non-NMDA) glutamate receptors. Longer latency responses were a mix of excitatory and inhibitory currents including non-NMDA EPSCs and GABAa receptor-mediated currents (IPSC). All synaptic responses exhibited prominent frequency-dependent depression. In a subset of neurons, we labeled sensory boutons by the anterograde fluorescent tracer, DiA, from aortic nerve baroreceptors and then recorded from anatomically identified second-order neurons. In identified second-order NTS neurons, ST activation evoked EPSCs with short to moderate latency (1.9-4.8 ms) but uniformly minimal jitter (31 to 61 micros) that were mediated by non-NMDA receptors but had failure rates as high as 39%. These monosynaptic EPSCs in identified second-order neurons were significantly different in latency and jitter than GABAergic IPSCs (latency, 2.95 +/- 0.71 vs. 5.56 +/- 0.74 ms, mean +/- SE, P = 0.027; jitter, 42.3 +/- 6.5 vs. 416.3 +/- 94.4 micros, P = 0.013, n = 4, 6, respectively), but failure rates were similar (27.8 +/- 9.0 vs. 9.7 +/- 4.4%, P = 0.08, respectively). Such results suggest that jitter and not absolute latency or failure rate is the most reliable discriminator of mono- versus polysynaptic pathways. The results suggest that brain stem sensory pathways may differ in their principles of integration compared with cortical models and that this importantly impacts synaptic performance. The unique performance properties of the sensory-NTS pathway may reflect stronger axosomatic synaptic processing in brain stem compared with dendritically weighted models typical in cortical structures and thus may reflect very different strategies of spatio-temporal integration in this NTS region and for autonomic regulation.
A unique text, relating the dramatic advances of modern neurobiology to the understanding of the structure and function of the autonomic nervous system. Written by an international group of distinguished scientists, it provides a clear view of the central neuronal components involved in autonomic control. Its scope is wide, ranging from anatomical pathways and molecular pharmacology to the perceptual qualities of autonomic sensation and their potential in modifying behaviour. The approach is mildly didactic, and combines concise background information with discussion of the latest research findings. It is richly illustrated with line drawings that concisely summarize concepts presented in the text.
The nucleus of the solitary tract (NTS) is the first location within the central nervous system for the integration and modulation of cardiovascular afferent as well as other viscerosensory input. The NTS is therefore a pivotal structure for maintaining homeostasis. This chapter examines the fundamental cellular and molecular building blocks of NTS pathways. It discusses NTS neurotransmitters, (glutamate and ϒ-amino-butyric acid, GABA) and the baroreceptor reflex, the mechanisms regulating afferent information transfer to sites beyond the NTS and the mechanisms by which two major modulators, angiotensin II and nitric oxide, transform afferent information related to cardiovascular regulation. Particular consideration is given to emerging views on the nature and role of heterogeneity in afferents and NTS neurons and their projection targets outside the NTS. The chapter also considers the impact of new signaling molecules in the endothelial interface between the bloodstream and brain on neural control of the circulation. © Oxford University Press, Inc. 2011, 1990. All rights reserved.
Key points
Successful transmission of information to the brain relies on a balance of excitation and inhibition, and this balance is different across different brain regions.
In the brain areas responsible for initiating homeostatic reflex control of visceral organs, excitation is known to be particularly powerful whereas inhibition is often less obvious.
Using minimal focal activation of single axons, this study shows that elementary inhibitory transmission in this brainstem region is founded on an intrinsically weak process with limited transmitter release that is surprisingly prone to failures.
Strong inhibitory transmission requires multi‐axon convergence of modestly reliable synapses, whereas primary afferent excitation arises from single, multi‐contact axons with highly reliable neurotransmitter release.
The results suggest that the entry of primary afferent information along cranial nerves enjoys a high safety factor in part due to the fundamental weakness of inhibitory transmission at these initial central neurons.
Abstract Visceral primary afferents enter the CNS at the caudal solitary tract nucleus (NTS), and activate central pathways key to autonomic and homeostatic regulation. Excitatory transmission from primary solitary tract (ST)‐afferents consists of multiple contacts originating from single axons that offer a remarkably high probability of glutamate release and high safety factor for ST afferent excitation. ST afferent activation sometimes triggers polysynaptic GABAergic circuits, which feedback onto second‐order NTS neurons. Although inhibitory transmission is observed at second‐order neurons, much less is known about the organization and mechanisms regulating GABA transmission. Here, we used a focal pipette to deliver minimal stimulus shocks near second‐order NTS neurons in rat brainstem slices and directly activated single GABAergic axons. Most minimal focal shocks activated low jitter EPSCs from single axons with characteristics resembling ST afferents. Much less commonly (9% of sites), minimal focal shocks activated monosynaptic IPSCs at fixed latency (low jitter) that often failed (30%) and had no frequency‐dependent facilitation or depression. These GABA release characteristics contrasted markedly to the unfailing, large amplitudes for glutamate released during ST‐EPCSs recorded from the same neurons. Surprisingly, unitary GABAergic IPSCs were only weakly calcium dependent. In some neurons, strong focal shocks evoked compound IPSCs indicating convergent summation of multiple inhibitory axons. Our studies demonstrate that second‐order NTS neurons receive GABAergic transmission from a diffuse network of inhibitory axons that rely on an intrinsically less reliable and substantially weaker release apparatus than ST excitation. Effective inhibition depends on co‐activation of convergent inputs to blunt excitatory drive.
The present series of experiments was designed to study the organization of preterminai processes and synaptic boutons of single physiologically identified slowly adapting receptor (SAR) pulmonary stretch afferent fibers. Intra‐axonally injected horseradish peroxidase‐wheat germ agglutinin (HRP‐WGA) conjugate was used as the label. In the first paper, we describe the pattern of arborization of axon collaterals from single physiologically identified SAR afferent fibers evident in the various subnuclei of the nucleus of the tractus solitarius (nTS). In the second paper, details are presented regarding the ultrastructure of these synaptic boutons and axon collaterals.
A number of significant findings resulted from this study: (1) A single lung stretch SAR afferent fiber arborized over a considerable distance rostrocaudally in the brain stem (1,700–2,100 μm). (2) A single lung stretch SAR afferent fiber terminated as hundreds of bouton terminals (650–1,180). (3) There was a remarkable consistency in the subnuclei of the nTS that received these terminal arborizations of SAR afferents. (4) The ventral (vnTS), intermediate (nI), ventrolateral (vInTS), and interstitial (ni) subnuclei of the nTS were the only regions of the nTS receiving bouton terminals of SAR afferent fibers. (5) Under the light microscope the pattern of termination of SAR afferents was similar in all the axons studied in this series. (6) The injected parent axon in each case could be followed in the TS at all levels and remained consistent with regard to position and orientation and could be traced rostrally to levels as far as 3.5 mm rostral to the obex whereas the region of terminal arborization was located around 1.7–2.1 mm rostral to the obex. This pattern indicates that a single lung stretch SAR afferent fiber descends caudally upon entering the nTS. In the cat vagal afferent fibers are known to enter the medulla at levels between 0.5 mm and 3.2 mm rostral to the obex (Kalia and Mesulam, '80a).
The results of the light microscopic analysis presented in this article indicate that lung stretch (SAR) afferents from the lungs and tracheobronchial tree have distinctly localized patterns of distribution in the nTS. In addition, these findings support the concept that representation of pulmonary afferents in the medulla is constituted by a differentiated distribution of nerve terminals in the various subnuclei of the nTS. Modality‐specific localization (SAR afferents in this case) appears to be predominant in the n'1'S. The widespread rostrocaudal distribution of the terminal field of a single lung stretch SAR afferent is remarkable. In addition, the finding that the terminals of different SAR afferents are localized in only a few subnuclei of the nTS suggests that specificity exists in this afferent system which could provide the morphological substrate for focussed physiological effects.
The nucleus of the solitary tract (NTS) contains a unique subpopulation of aldosterone-sensitive neurons. These neurons express the enzyme 11-β-hydroxysteroid dehydrogenase type 2 (HSD2) and are activated by sodium deprivation. They are located in the caudal NTS, a region which is densely innervated by the vagus nerve, suggesting that they could receive direct viscerosensory input from the periphery. To test this possibility, we injected the highly sensitive axonal tracer biotinylated dextran amine (BDA) into the left nodose ganglion in rats. Using confocal microscopy, we observed a sparse input from the vagus to most HSD2 neurons. Roughly 80% of the ipsilateral HSD2 neurons exhibited at least one close contact with a BDA-labeled vagal bouton, although most of these cells received only a few total contacts. Most of these contacts were axo-dendritic (∼ 80%), while ∼ 20% were axo-somatic. In contrast, the synaptic vesicular transporters VGLUT2 or GAD7 labeled much larger populations of boutons contacting HSD2-labeled dendrites and somata, suggesting that direct input from the vagus may only account for a minority of the information integrated by these neurons. In summary, the aldosterone-sensitive HSD2 neurons in the NTS appear to receive a small amount of direct viscerosensory input from the vagus nerve. The peripheral sites of origin and functional significance of this projection remain unknown. Combined with previously-identified central sources of input to these cells, the present finding indicates that the HSD2 neurons integrate humoral information with input from a variety of neural afferents.
The anterograde fluorescent tracer DiA was used to visualize baroreceptor fibers and synaptic terminals both in living and fixed tissue. Baroreceptor fibers labeled with DiA terminated as a dense synaptic field in the medial nucleus tractus solitarius (NTS), making synaptic contact on the soma, as well as processes of neurons that they innervated. A similar distribution and morphology was observed in baroreceptor fibers and terminals labeled with horseradish peroxidase. DiA also identified baroreceptor terminals and the neurons receiving these synaptic contacts in vitro. NTS neurons were dissociated from their surrounding tissue and identified by attached baroreceptor terminals that retained the fluorescent dye. These results will enable us to study the electrophysiological properties of dispersed neurons that receive identified baroreceptor synaptic terminals.
The connectivity diagram of neocortical circuits is still unknown, and there are conflicting data as to whether cortical neurons are wired specifically or not. To investigate the basic structure of cortical microcircuits, we use a two-photon photostimulation technique that enables the systematic mapping of synaptic connections with single-cell resolution. We map the inhibitory connectivity between upper layers somatostatin-positive GABAergic interneurons and pyramidal cells in mouse frontal cortex. Most, and sometimes all, inhibitory neurons are locally connected to every sampled pyramidal cell. This dense inhibitory connectivity is found at both young and mature developmental ages. Inhibitory innervation of neighboring pyramidal cells is similar, regardless of whether they are connected among themselves or not. We conclude that local inhibitory connectivity is promiscuous, does not form subnetworks, and can approach the theoretical limit of a completely connected synaptic matrix.
Astrocytes are now considered as essential partners of neurons. In particular, they play important roles in glutamatergic transmission, including transmitter inactivation by uptake. Here, we investigated the organization of astroglia in the Nucleus Tractus Solitarii (NTS), a sensory nucleus located in the caudal medulla. Special attention was given to perisynaptic astroglial processes. Investigations were performed at the light and electron microscope levels, using immunodetection of glial glutamate transporters, stereological methods, and serial reconstruction. In the NTS, the main glutamate transporter expressed by astrocytes was GLT1. The volume fraction of astrocyte processes and the density of astrocyte membranes reached 15% and 2.8 μm(2) μm(-3) , respectively. In spite of the relative abundance of astrocyte processes, we found that NTS glutamatergic synapses were not entirely surrounded by glia. Measurements were performed on 43 reconstructed asymmetric junctions which were either single synapses (n = 22) or synapses involved in multisynaptic arrangements (n = 21). Single synapses had 58% of their perimeter contacted by astrocyte processes on average. In multisynaptic arrangement, glial coverage was restricted to the outer part of synaptic diameters and amounted to 50% of this outer part on average. Incomplete glial coverage of NTS synapses may allow glutamate to diffuse out of the synaptic cleft and to activate extrasynaptic receptors as well as receptors from neighboring synapses. Especially, in multisynaptic arrangements, the lack of intervening glia may favor functional coupling between individual contacts.
Synaptic terminals often contain metabotropic receptors that act as autoreceptors to control neurotransmitter release. Less appreciated is the heterosynaptic crossover of glutamate receptors to control GABA release and vice versa GABA receptors which control glutamate release. In the brainstem, activation of solitary tract (ST) afferents releases glutamate onto second-order neurons within the solitary tract nucleus (NTS). Multiple metabotropic receptors are expressed in NTS for glutamate (mGluRs) and for GABA (GABA(B)). The present report identifies mGluR regulation of glutamate release at second and higher order sensory neurons in NTS slices. We found strong inhibition of glutamate release to group II and III mGluR activation on mechanically isolated NTS neurons. However, the same mGluR-selective antagonists paradoxically decreased glutamate release (miniature, mEPSCs) at identified second-order NTS neurons. Unaltered amplitudes were consistent with selective presynaptic mGluR actions. GABA(B) blockade in slices resolved the paradoxical differences and revealed a group II/III mGluR negative feedback of mEPSC frequency similar to isolated neurons. Thus, the balance of glutamate control is tipped by mGluR receptors on GABA terminals resulting in predominating heterosynaptic GABA(B) inhibition of glutamate release. Regulation by mGluR or GABA(B) was not consistently evident in excitatory postsynaptic currents (EPSCs) in higher-order NTS neurons demonstrating metabotropic receptor distinctions in processing at different NTS pathway stages. These cellular localizations may figure importantly in understanding interventions such as brain-penetrant compounds or microinjections. We conclude that afferent glutamate release in NTS produces a coordinate presynaptic activation of co-localized mGluR and GABA(B) feedback on cranial afferent terminals to regulate glutamate release.
TRPV1 receptors feature prominently in nociception of spinal primary afferents but are also expressed in unmyelinated cranial visceral primary afferents linked to homeostatic regulation. Cranial visceral afferents enter the brain at the solitary tract nucleus (NTS) to control the heart, lungs, and other vital organs. Here we identify a role for central TRPV1 in the activity-dependent facilitation of glutamatergic transmission from solitary tract (ST) afferents. Fast, synchronous ST-NTS transmission from capsaicin-sensitive (TRPV1+) and -insensitive (TRPV1-) afferents was similar. However, afferent activation triggered long-lasting asynchronous glutamate release only from TRPV1+ synapses. Asynchronous release was proportional to synchronous EPSC amplitude, activity, and calcium entry. TRPV1 antagonists and low temperature blocked asynchronous release, but not evoked EPSCs. At physiological afferent frequencies, asynchronous release strongly potentiated the duration of postsynaptic spiking. This activity-dependent TPRV1-mediated facilitation is a form of synaptic plasticity that brings a unique central integrative feature to the CNS and autonomic regulation.
The HSD2 (11-beta-hydroxysteroid dehydrogenase-type 2 enzyme) containing neurons of the nucleus tractus solitarius (NTS) become activated during low-sodium and high-aldosterone states such as hypovolemia. This response may be due to hormonal and/or neural signals. Hormonal signals may activate neurons in the area postrema that innervate the HSD2 neurons. The vagus nerve projects directly to the HSD2 neurons and this could be another route whereby these neurons receive information about systemic sodium/aldosterone status. The peripheral sites of origin that contribute to this vagal projection remain unknown, and in the present study, we injected the transganglionic tracer, cholera toxin beta-subunit-horseradish peroxidase (CTb-HRP), into wall of various gastrointestinal organs (stomach, small and large intestine) or liver of rats. Confocal microscopy of brainstem sections stained by a double immunohistochemical procedure was used to analyze whether the HSD2 neurons received axonal contacts from specific gastrointestinal structures. The major source of afferents arose from the stomach, mainly from its pyloric antrum, but a weaker input originated from the fundus region. A trace amount originated from the duodenum. The terminal part of the small intestine and large intestine did not to contribute to this projection. Similarly, no afferent inputs from the liver or portal vein were found. In conclusion, HSD2 neurons receive an input mainly from the stomach and these results are considered as potential sites affecting sodium intake.
For over a century, electrical microstimulation has been the most direct method for causally linking brain function with behavior. Despite this long history, it is still unclear how the activity of neural populations is affected by stimulation. For example, there is still no consensus on where activated cells lie or on the extent to which neural processes such as passing axons near the electrode are also activated. Past studies of this question have proven difficult because microstimulation interferes with electrophysiological recordings, which in any case provide only coarse information about the location of activated cells. We used two-photon calcium imaging, an optical method, to circumvent these hurdles. We found that microstimulation sparsely activates neurons around the electrode, sometimes as far as millimeters away, even at low currents. Our results indicate that the pattern of activated neurons likely arises from the direct activation of axons in a volume tens of microns in diameter.
It is now well established that brain plasticity is an inherent property not only of the developing but also of the adult brain. Numerous beneficial effects of exercise, including improved memory, cognitive function and neuroprotection, have been shown to involve an important neuroplastic component. However, whether major adaptive cardiovascular adjustments during exercise, needed to ensure proper blood perfusion of peripheral tissues, also require brain neuroplasticity, is presently unknown. This review will critically evaluate current knowledge on proposed mechanisms that are likely to underlie the continuous resetting of baroreflex control of heart rate during/after exercise and following exercise training. Accumulating evidence indicates that not only somatosensory afferents (conveyed by skeletal muscle receptors, baroreceptors and/or cardiopulmonary receptors) but also projections arising from central command neurons (in particular, peptidergic hypothalamic pre-autonomic neurons) converge into the nucleus tractus solitarii (NTS) in the dorsal brainstem, to co-ordinate complex cardiovascular adaptations during dynamic exercise. This review focuses in particular on a reciprocally interconnected network between the NTS and the hypothalamic paraventricular nucleus (PVN), which is proposed to act as a pivotal anatomical and functional substrate underlying integrative feedforward and feedback cardiovascular adjustments during exercise. Recent findings supporting neuroplastic adaptive changes within the NTS-PVN reciprocal network (e.g. remodelling of afferent inputs, structural and functional neuronal plasticity and changes in neurotransmitter content) will be discussed within the context of their role as important underlying cellular mechanisms supporting the tonic activation and improved efficacy of these central pathways in response to circulatory demand at rest and during exercise, both in sedentary and in trained individuals. We hope this review will stimulate more comprehensive studies aimed at understanding cellular and molecular mechanisms within CNS neuronal networks that contribute to exercise-induced neuroplasticity and cardiovascular adjustments.
Inhibitory connectivity onto neocortical pyramidal cells was mapped using LSPS (laser-scanning photostimulation/glutamate uncaging). The average onset latency of IPSCs was shorter than that of EPSCs recorded in the same cells, indicating a specific mechanism for rapid network recruitment of inhibition. The majority of strong inhibitory synaptic inputs originated within 300 mum of the recorded cell's soma, had onset latencies between 4 and 10 ms, and high amplitude [short-latency IPSCs (slIPSCs)]. slIPSCs were GABA(A) receptor- mediated chloride currents that were evoked in an all-or-none manner. We tested whether slIPSCs resulted from somatic depolarization of presynaptic interneurons or from direct excitation of inhibitory presynaptic terminals via kainate receptors. Our evidence supports the former hypothesis: (1) slIPSCs had similar sensitivity to kainate and AMPA receptor blockers as electrically evoked EPSCs. (2) slIPSCs frequently had an notched rising phase suggestive of summated IPSCs resulting from repetitive firing of presynaptic neurons. (3) Latencies and interevent intervals were consistent with spike latencies and interspike intervals in fast-spiking (FS) interneurons. (4) slIPSCs were frequently evoked at spots where the recorded cell was also excited directly, but approximately 15% of spots from which slIPSCs were evoked did not overlap with the recorded neuron's cell body. We propose that slIPSCs from FS interneurons represent a pool of powerful inhibitory signals that can be recruited by local excitation. Because of their magnitude, progressive recruitment, and short latency, slIPSCs are a effective mechanism of regulating excitability in neocortical circuits.
Cranial nerve visceral afferents enter the brain stem to synapse on neurons within the solitary tract nucleus (NTS). The broad heterogeneity of both visceral afferents and NTS neurons makes understanding afferent synaptic transmission particularly challenging. To study a specific subgroup of second-order neurons in medial NTS, we anterogradely labeled arterial baroreceptor afferents of the aortic depressor nerve (ADN) with lipophilic fluorescent tracer (i.e., ADN+) and measured synaptic responses to solitary tract (ST) activation recorded from dye-identified neurons in medial NTS in horizontal brain stem slices. Every ADN+ NTS neuron received constant-latency ST-evoked excitatory postsynaptic currents (EPSCs) (jitter < 192 micros, SD of latency). Stimulus-recruitment profiles showed single thresholds and no suprathreshold recruitment, findings consistent with EPSCs arising from a single, branched afferent axon. Frequency-dependent depression of ADN+ EPSCs averaged approximately 70% for five shocks at 50 Hz, but single-shock failure rates did not exceed 4%. Whether adjacent ADN- or those from unlabeled animals, other second-order NTS neurons (jitters < 200 micros) had ST transmission properties indistinguishable from ADN+. Capsaicin (CAP; 100 nM) blocked ST transmission in some neurons. CAP-sensitive ST-EPSCs were smaller and failed over five times more frequently than CAP-resistant responses, whether ADN+ or from unlabeled animals. Variance-mean analysis of ST-EPSCs suggested uniformly high probabilities for quantal glutamate release across second-order neurons. While amplitude differences may reflect different numbers of contacts, higher frequency-dependent failure rates in CAP-sensitive ST-EPSCs may arise from subtype-specific differences in afferent axon properties. Thus afferent transmission within medial NTS differed by axon class (e.g., CAP sensitive) but was indistinguishable by source of axon (e.g., baroreceptor vs. nonbaroreceptor).
The ultrastructure of fibres and sensory terminals of the aortic nerve innervating the aorta between the left common carotid and left subclavian arteries was investigated in the rat. This is the region from which most baroreceptor responses are recorded electrophysiologically. The fibres of the aortic nerve enter the adventitia and separate into bundles generally containing one myelinated fibre and four or five unmyelinated fibres of various sizes. The bundles pursue a roughly helical course through the adventitia; when they are close to the aortic media, the myelinated fibre loses its myelin sheath. A complex sensory terminal region is formed, as both the unmyelinated and 'premyelinated' axons become irregularly varicose. The concentration of mitochondria becomes very dense and cytoplasmic deposits of glycogen are observed. Both unmyelinated and premyelinated axons branch, and the unmyelinated axons wind irregularly around the premyelinated axon. The latter may have several loops and small holes. The terminal regions of both types of axon contain clusters of clear 40 nm vesicles. Part of the surface of each terminal region is ensheathed by Schwann cells, but the rest of the axolemma is directly exposed to extracellular connective tissue. There are often several layers of basal lamina around the sensory terminals and parts of the axolemma and Schwann cell membranes are attached to it by fine fibrillar material. The basal laminae are also attached to fibroblasts, fibroblast-like perineurial cells and elastic laminae, and the whole cellular and extracellular system appears to be tightly bound together. No differences between baroreceptors of spontaneously hypertensive and normal rats were found.
Indirect evidence suggests that excitatory amino acids (EAA) are involved in synaptic transmission of visceral afferents at their synapses within the nucleus tractus solitarius (NTS). Little is known about the identity of the postsynaptic receptors or response mechanisms. Here we report results from a longitudinal brain slice of the rat medulla. Intracellular recordings were made from neurons in delimited portions of the dorsal medial NTS (mNTS) known to receive baroreceptor inputs. Stimulation of the solitary tract 1-3 mm from the mNTS recording site evoked short (2 ms) latency excitatory postsynaptic potentials (EPSPs), which had durations of 40-50 ms. Addition of the non-N-methyl-D-aspartate (non-NMDA) selective antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) to the slice surface near the recording electrode resulted in a rapid (within 30-45 s) suppression of the EPSP. Complete EPSP blockade was only slowly reversed by drug-free saline. Concentration-response relations (n = 14) showed 50% depression of EPSPs by surface concentrations of 1-10 microM CNQX. EPSP amplitude was resistant to the selective NMDA antagonist 2-amino-5-phosphonovalerate (AP 5) and, on average, was reduced less than 20% at 100 microM AP 5, an effect that was not statistically significant (n = 10; P greater than 0.05). In conclusion, this study offers the first direct evidence that EAAs mediate the primary events of afferent synaptic transmission in NTS. The experiments suggest that excitatory sensory afferent synaptic transmission to mNTS neurons is mediated by an EAA transmitter acting at non-NMDA receptors, but NMDA receptors may have a modulatory role.
The organization of axon collaterals, preterminal processes, and presumptive synaptic boutons of single physiologically identified rapidly adapting receptor (RAR) pulmonary afferent fibers was examined following the intraaxonal application of wheat germ agglutinin conjugated with horseradish peroxidase (WGA‐HRP). The RAR axons were injected 200–300 μm lateral to the nucleus of the tractus solitarius (nTS) at a number of different rostrocaudal levels in seven individual experiments. The trajectories of the stained axons were reconstructed from individual 50‐μm‐thick serial sections. The rostrocaudal extent, as well as the distribution of the trajectory of each RAR afferent, was reconstructed from every section by using a camera lucida attachment. In this first of two papers, we describe the pattern of organization of bouton terminals of RAR afferents related to cytoarchitectonically distinct subnuclei of the nTS. In the companion paper, morphological details of the fine structure of these synaptic boutons and axonal branches are described in different subnuclei in order to illustrate morphological differences in these functionally distinct regions.
A number of significant findings have resulted from this light microscopic study. The central process of a single RAR afferent fiber arborized in the medulla oblongata over a considerable distance in the rostrocaudal plane (2.5 mm rostral to 1.4 mm caudal to the obex). A single RAR afferent fiber terminated in numerous bouton terminals (range 500–1,050), and these terminals arose from over 400 segments of branches of the parent injected axon. A small number of en passant bouton terminals were found. There appeared to be a remarkable degree of consistency in the subnuclei of the nTS where these terminals arborized. The dorsal and dorsolateral subnuclei of the nTS received 144–647 bouton terminals. The second‐largest concentration of bouton terminals of RAR afferents was found in the intermediate (nI) subnucleus of the nTS. No labeled bouton terminal was found in the ventral and ventrolateral subnuclei of the nTS. This finding is in sharp contrast to the terminations of SAR afferents which terminated predominantly in the ventral and ventrolateral nuclei of the nTS, the interstitial nucleus of the nTS, and the nI.
The parent RAR axon could be traced as far rostrally as 2.5 mm, even though the region of terminal arborization could not be followed beyond 0.8 mm. The destination of this rostrally projecting RAR afferent could not be determined in this study. This pattern indicates that a single RAR afferent fiber ascends rostrally in the tractus solitarius giving off branches at a number of different levels.
Since the rostrocaudal location of the randomly selected RAR afferent impaled in the medulla varied considerably in all seven cases examined, it is likely the location of their peripheral endings was different in each case. In spite of this viscerotopic difference between the injected axons, the patern of distribution of the axon collaterals and synaptic boutons in the nTS was very similar in all cases.
This illustrates that modality specificity, and not viscerotopic specificity, is an important feature of the central organization of these afferents. These findings are similar to the modality specificity observed with SAR afferents (Kalia and Richter: J. Comp. Neurol. 233 : 308‐332, '85a). In addition, these results further support the concept of functional organization of the nTS along cytoarchitectonic boundaries.
Post-synaptic responses evoked in neurones of the nucleus tractus solitarius by electrical stimulation of the carotid sinus, aortic and vagal nerves, alone or in combination, have been studied in anaesthetized cats using both extracellular and intracellular recording techniques. A total of 292 neurones received an input from at least one of the three nerves tested. The activity of the large majority of these cells (249) could only be shown to be altered by stimulation of one of these nerves and in 222 of these cases this was an excitatory response. These responses showed the expected post-synaptic characteristics including temporal summation and, in intracellular records, a summation of evoked excitatory post-synaptic potentials (e.p.s.p.s). The minimum latency to onset of these responses was variable, both for individual cells and for the population as a whole and varied within the range 2-124 ms. In a small number of cells (twenty-seven), the input was purely inhibitory in nature. In neurones showing a tonic discharge this produced a decrease in the rate of firing. This influence was most marked in intracellular records where membrane hyperpolarizations were noted. Again, the latency to onset was variable, in the range 4-27 ms. Convergent inputs from two or more of the nerves were identified in forty-three neurones. The effects of these were always excitatory. They could be observed both as a facilitation of spike activity recorded extracellularly and as summation of subliminally evoked e.p.s.p.s recorded intracellularly. On the basis of threshold voltages and latency to onset, the afferents to these neurones are indistinguishable from those providing an exclusive input. It can be concluded that at least some of the neurones in the nucleus tractus solitarius and its vicinity receive inputs from more than one source. The implications of these observations on the role of this brain-stem area in cardiorespiratory reflexes is discussed.
The nucleus of the tractus solitarius is a site for termination of primary afferents originating from a variety of visceral receptors. The localization of bouton terminals of slowly adapting lung stretch (SAR) afferent fibers originating from the tracheobronchial tree have been described in the companion paper (Kalia and Richter, '85). The most conspicuous finding regarding the location of SAR terminals is that they are concentrated within specific subnuclear groups of the nucleus of the tractus solitarius (nTS) and are distributed widely in the rostrocaudal plane of the medulla oblongata. These light microscopic features have provided us with valuable information with regard to the organization of visceral afferents in the central nervous system. The synaptic profiles formed by the 476 bouton terminals of these HRP-labeled afferents have been described in this paper in serial thin sections. All of the bouton terminals examined under the electron microscope were found to contain round synaptic vesicles. Synaptic boutons (1.0–3.0) /μm in diameter were usually of the en passant variety and made contact with different structures depending upon the subnucleus which was examined. In the ventral (v) and the ventrolateral (vl) subnuclei of the nTS, asymmetrical (type I) synaptic contacts containing round, clear synaptic vesicles of 35–50 ion μm in diameter were found and these contacts were made with (1) the soma of cell bodies located in that subnucleus; (2) spiny dendrites in that nucleus; (3) vesicle-containing axon terminals that were presynaptic to the HRP-labeled bouton terminal; and (4) vesicle-containing dendrites in which the HRP profile was presynaptically located. The terminal axon remained my-elinated till the last 1 μm before the bouton terminal was formed. There was no distinct, unmyelinated portion of the terminal axon. The synaptic bouton received axon-axonal synapses from unlabeled bouton terminals containing round, clear vesicles.
The three-dimensional branching pattern and ultrastructure of afferent myelinated fibers and their terminals located in the trachealis muscle of the dog are described. The afferent endings are believed to be those of the slowly adapting stretch receptors of the trachea. They have structural features typical of mechanoreceptors: distal to the loss of myelin, their shape becomes more irregular and the cytoplasm is filled with mitochondria, glycogen, and osmiophilic bodies. In some places the cell membrane is attached directly to basal lamina without interposition of a Schwann cell. A bundle of unmyelinated fibers accompanies each myelinated fiber and continues for an undetermined distance beyond (luminal to) terminations of the myelinated fiber. The unmyelinated fibers contain many round, clear vesicles and a few dense-cored vesicles and are also attached directly to basal lamina in places. Three-dimensional reconstruction of three receptors revealed three quite different branching patterns, but all included apparent rings as part of more or less contorted terminal regions (some neurons apparently having more than one terminal region). No obvious structural basis for the activation of receptors by transverse but not longitudinal stretch was found.
The medullary projections of afferent neurons with cell bodies in the petrosal ganglion have been investigated using an antidromic mapping technique. Of the ninety-three units studied, fifty-eight were shown to have patterns of discharge indicating that they were baroreceptors and thirty-five showed responses to stimuli indicating that they were arterial chemoreceptors. Twelve baroreceptor and thirteen chemoreceptor afferents had sufficiently stable unitary discharges to permit a detailed estimation of some of their central projections using stimulation through monopolar tungsten micro-electrodes to evoke antidromic spikes. In order to estimate their pattern of projection, depth-threshold contours for each penetration through the dorsomedial medulla and the values of antidromic latency were considered. Baroreceptor afferent fibres with myelinated (six units) and non-myelinated (six units) axons showed similar patterns of central projection. All could be activated from the ipsilateral nucleus of the tractus solitarius (n.t.s.), most often from its lateral divisions rostral to the obex. The dorsolateral and dorsomedial portions of the n.t.s. were most often innervated, with the commissural subnucleus receiving an innervation in seven of the twelve neurones studied. Stimulation of the ventrolateral subnucleus was effective in activating two afferent fibres whilst stimulation of the ventral subnucleus was effective in only one case. All chemoreceptor afferent fibres had calculated conduction velocities less than 4 m/s and all were activated from the dorsomedial and medial subnuclei of the ipsilateral n.t.s. In twelve of the thirteen neurones investigated in detail there was evidence of an innervation of the commissural nucleus both at the level of the obex and behind it. In three cases this extended into the contralateral portion of the commissural nucleus. In four cases a sparse innervation of the lateral subnucleus, comprising its dorsolateral aspects, was seen. The potential significance of these distinctive patterns of projection of arterial baroreceptors and chemoreceptors is discussed in relation to cardiovascular and respiratory control.
In anaesthetized cats, right cardiac vagal branches were electrically stimulated and recordings of evoked 'slow wave' and single neurone activity were made in the brain stem. Short-latency 'slow wave' and multi-neuronal activity evoked by excitation of myelinated vagal afferent fibres were recorded in the medial and lateral subnuclei of the nucleus tractus solitarius, the area postrema, the dorsal vagal motor nucleus, the lateral reticular formation and the nucleus ambiguus. Long-latency responses evoked by vagal non-myelinated fibres were recorded in the medial subnucleus of the nucleus tractus solitarius, the area postrema, dorsal vagal motor nucleus, the parahypoglossal area and the lateral reticular formation dorsal to the nucleus ambiguus. A specific study was made of seventy-two single neurones activated by non-myelinated afferent fibres in the cardiac branch. Thirty-four were shown to be synaptically activated, twenty-one were activated nonsynaptically and seventeen could not be classified. One neurone was also activated by myelinated cardiac afferent fibres, and two by thoracic vagal (including pulmonary) afferent fibres. Neurones were not spontaneously active. Indirect evidence suggests that the majority of the recordings of nonsynaptically activated neurones were likely to be from cell bodies. Neurones were located from the level of the obex to 3.0 mm rostral to it in the medial subnucleus of the nucleus tractus solitarius (45), and in the lateral subnucleus (2), the area postrema and its border with the medial subnucleus of the nucleus tractus solitarius (13), the dorsal vagal motor nucleus (9), the parahypoglossal area (1) and the lateral reticular formation dorsal to nucleus ambiguus (2). Recordings were made from fifteen neurones activated by myelinated fibres in the cardiac vagal branches, and twelve were excited synaptically. The neurones were located in the medial (8) and lateral (3) subnuclei of the nucleus tractus solitarius, the dorsal vagal motor nucleus (1) and the lateral reticular formation (1). Four neurones were also excited by vagal afferent fibres in the thoracic vagal nerve immediately caudal to the caudal cardiac branch.
1. Micro-electrode recordings were made from slowly adapting pulmonary stretch afferents within the nodose ganglia of cats and rabbits. Recordings sites were distributed throughout the ganglia. 2. The projections of these afferents to the medulla oblongata were studied by antidromic stimulation. 'Point' and 'Field' type depth--threshold curves were interpreted as corresponding to stimulation of the main afferent axons and its branches, respectively. Increases in antidromic latency in conjunction with 'field' contours was additional evidence in support of this interpretation. 3. In cats, most (six out of seven) afferents had extensive branches, and probably also terminations, within the medial subnucleus of the ipsilateral nucleus tractus solitarius (n.t.s.). Two of these, plus one other afferent, also had projections to the lateral and ventrolateral subnuclei. 4. In rabbits the projections of such afferents were similar, i.e. mainly to the medial subnucleus of the n.t.s. (eight out of eleven) but also extending into the nucleus alaris, and occasionally to lateral and ventrolateral subnuclei (two out of eleven) or to both regions (one out of eleven). 5. Branching of single afferents was seen to occur over up to 3 mm of the rostro-caudal extent of the intermediate region of the n.t.s. The significance of the observations is discussed.
1. Neurons of the nodose ganglia provide the sole connection between many types of visceral sensory inputs and the central nervous system. Electrophysiological studies of isolated nodose neurons provide a practical means of measuring individual cell membrane currents and assessing their putative contributions to the overall response properties of the neuron and its terminations. Here, we present a comprehensive mathematical model of an isolated nodose sensory neuron that is based upon numerical fits to quantitative voltage- and current-clamp data recorded in our laboratory. Model development was accomplished using an iterative process of electrophysiological recordings, nonlinear parameter estimation, and computer simulation. This work is part of an integrative effort aimed at identifying and characterizing the fundamental ionic mechanisms participating in the afferent neuronal limb of the baroreceptor reflex. 2. The neuronal model consists of two parts: a Hodgkin-Huxley-type membrane model coupled to a lumped fluid compartment model that describes Ca2+ ion concentration dynamics within the intracellular and external perineuronal media. Calcium buffering via a calmodulin-type buffer is provided within the intracellular compartment. 3. The complete model accurately reproduces whole-cell voltage-clamp recordings of the major ion channel currents observed in enzymatically dispersed nodose sensory neurons. Specifically, two Na+ currents exhibiting fast (INaf) and slow tetrodotoxin (TTX)-insensitive (INas) kinetics; low- and high-threshold Ca2+ currents exhibiting transient (ICa,t) and long-lasting (ICa,n) dynamics, respectively; and outward K+ currents consisting of a delayed-rectifier current (IK), a transient outward current (I(t)) and a Ca(2+)-activated K+ current (IK,Ca). 4. Whole-cell current-clamp recordings of somatic action-potential dynamics were performed on enzymatically dispersed nodose neurons using the perforated patch-clamp technique. Stimulus protocols consisted of both short (< or = 2.0 ms) and long (> or = 200 ms) duration current pulses over a wide range of membrane holding potentials. These studies clearly revealed two populations of nodose neurons, often termed A- and C-type cells, which exhibit markedly different action-potential signatures and stimulus response properties. 5. Using a single set of equations, the model accurately reproduces the electrical behavior of both A- and C-type nodose neurons in response to a wide variety of stimulus conditions and membrane holding potentials. The structure of the model, as well as the majority of its parameters are the same for both A- and C-type implementations.(ABSTRACT TRUNCATED AT 400 WORDS)
We demonstrated the convergence of information from the pharyngeal and laryngeal mucosa, transmitted by the glossopharyngeal nerve (GPN) and superior laryngeal nerve (SLN), in the nucleus of the tractus solitarius (NTS). First, the distribution of terminals of the GPN and SLN in the NTS was examined by an HPR tracing technique in cats, and the synapse formation of these neurons with NTS neurons was demonstrated by electron microscopy. The HRP-labeled SLN and GPN terminals were localized in a small area of the interstitial subnucleus of the NTS, slightly rostral to the obex, forming synapses with NTS neurons. Next, using extracellular recording in anesthetized cats, we determined whether or not swallowing-related neurons in the medulla oblongata receive peripheral inputs. Convergence of peripheral sensory inputs from the SLN and GPN was observed in more than 80% of the NTS cells. These results suggest that the NTS is not only a sensory-relay nucleus but also integrates information necessary for eliciting protective reflexes of the upper airway, such as swallowing.
A new technique for understanding the organization of complex circuits in the vertebrate brain, scanning laser photostimulation, is described. This approach is based on the photolysis of a caged form of the excitatory neurotransmitter glutamate. Computer-controlled photostimulation and whole cell recording in brain slices allow the construction of detailed maps of the position, strength, sign and number of inputs converging on a single postsynaptic neuron. Scanning laser photostimulation offers many advantages over current techniques: spatial resolution is superb, fibers of passage are not activated, and thousands of presynaptic locations can be stimulated. This review describes the technique of photostimulation, outlines the instrumentation, necessary to implement it, and discusses the interpretation of photostimulation-derived data. Several examples of applications, ranging from mapping circuits in the mammalian visual cortex to determining receptor distributions on single neurons are considered. Although still in its early stages, scanning laser photostimulation offers neuroscientists a powerful tool for determining the organization and function of local brain circuits.
Previous neuroanatomic and physiologic studies indicated that afferent fibres from slowly adapting pulmonary stretch receptors (SAR) project to the nuclei of the solitary tract and terminate on inspiratory beta-neurons. In the present study we combined electrophysiologic and morphologic approaches to verify the presumed monosynaptic connections between SARs and beta-neurons.
Single identified beta-neurons and single identified SAR afferent fibres were labelled intrasomally and intraaxonally, respectively, with horseradish peroxidase (HRP) in the same anesthetized cats. Under the light microscope, we analyzed the morphology of beta-neurons and their dendritic fields and of the terminal projection pattern of fibres from SARs and identified potential synaptic connections between boutons of SAR afferent fibres and the soma and dendrites of beta-neurons. The identified tissue was then processed further for electron microscopic analysis.
On average, beta-neurons had 6 primary dendrites that bifurcated 3–8 times. The dendritic trees extended 1.5 mm both rostrocaudally in the ventrolateral nucleus of the solitary tract and medially into the intermediate subnucleus. Axons of beta-neurons curved toward the midline and no collateral branches were evident over its stained length (2.5–3.4 mm).
Axodendritic synaptic contacts between SAR fibres and beta-neurons were identified electron microscopically in four of six tissue samples chosen by light microscopy. In addition, we located 2 axodendritic and 2 axosomatic synaptic contacts that were not observed under light microscopic screening. The boutons of SAR fibres contained clear, round vesicles and formed asymmetrical synapses with beta-neurons. Multiple synaptic connections were found between collaterals of a single SAR and single beta-neurons, indicating a dense terminal projection of single SAR afferent fibres onto beta-neurons. These morphologic data prove monosynaptic connections between electrophysiologically identified SAR afferent fibres and beta-neurons.
Area postrema neurons enhance baroreflex function, perhaps by augmenting baroreceptor afferent processing in the nucleus tractus solitarius (NTS). If so, NTS neurons should receive convergent excitatory inputs from area postrema neurons and baroreceptors. The aims of this study were to record extracellular activity of NTS neurons to determine whether 1) area postrema and aortic baroreceptor afferents converged in NTS, 2) area postrema and vagal afferents converged in NTS, and 3) the convergent inputs were facilitative. Studies were performed in pentobarbital sodium- or alpha-chloralose-anesthetized rabbits. Forty-six of 194 NTS neurons received inputs from the area postrema and aortic depressor nerve. Twelve of the 23 inputs showed facilitative summation; unit response rate evoked by paired inputs (79%) doubled the predicted (calculated) response rate for simple addition (37%). Fifty-eight of 114 NTS neurons received excitatory inputs from the area postrema and vagus. Eleven of the 13 inputs showed facilitative summation; unit response to paired inputs (87%) doubled the predicted response (44%). Area postrema neurons may augment the processing of aortic and vagal inputs by NTS neurons and, hence, enhance the reflex output of these afferent pathways.
A population of 43 neurons in the nucleus of the solitary tract (NTS) was identified in pentobarbital sodium anesthetized, paralyzed, and artificially ventilated cats that received convergent inputs from carotid sinus nerve (CSN) and superior laryngeal nerve (SLN) afferents. In 21 neurons, electrical stimulation of the CSN and SLN each evoked an excitatory postsynaptic potential (EPSP; mean onset latency +/- SE of CSN-evoked input = 7.2 +/- 0.8 ms, range 2.1-14.1 ms; of SLN-evoked input = 10.3 +/- 2.1 ms, range 2.8-46.8 ms). In 22 neurons, electrical stimulation of either the CSN or SLN evoked an EPSP/ inhibitory postsynaptic potential (IPSP) sequence (CSN-evoked input = 6.7 +/- 0.6 ms, range 2.1-12.2 ms; SLN-evoked input = 8.4 +/- 0.8 ms, range 3.0-19.4 ms). Spatial interactions (facilitation, summation, occlusion) and time-dependent inhibitory interactions were observed between the convergent inputs. Natural stimulation of specific receptors indicated that 14 cells received a convergent excitatory input from carotid sinus baroreceptors and laryngeal mechanoreceptors, 10 received a convergent excitatory input from carotid body chemoreceptors and laryngeal mechanoreceptors, and 5 received a convergent excitatory input from baroreceptors, chemoreceptors, and laryngeal mechanoreceptors. The interactions and various patterns of convergence suggest a significant integration of convergent inputs from disparate afferent sources by these neurons.
1. Temporal processing of heterogenous afferent signals by nucleus of the solitary tract (NTS) neurons has been previously characterized. Experiments were performed in 26 pentobarbital-sodium-anesthetized male Sprague-Dawley rats to characterize the temporal processing of evoked activity in NTS neurons with the use of the aortic nerve, which contains exclusively arterial baroreceptor afferent fibers. 2. Extracellular single-cell activity was examined in the NTS during electrical stimulation of the aortic nerve with the use of a conditioning-test paradigm. 3. Results were obtained from 49 neurons, 22 of which were characterized as receiving monosynaptic input from aortic nerve afferents. The average number of evoked potentials per aortic nerve stimulation was 1.1 +/- 0.1 (SE) for the monosynaptic neurons and 1.2 +/- 0.2 for the polysynaptic neurons. Spontaneous activity averaged 3.7 +/- 0.7 Hz. No neuron exhibited an obvious pulse-rhythmic discharge. The average peak onset latency for monosynaptic cells of 17 +/- 2 ms (range 3-31 ms) was significantly (P < 0.05) shorter than the average of 26 +/- 1 ms (range 13-38 ms) for the polysynaptic cells. The average onset latency variability was also less in monosynaptic compared with polysynaptic cells (4 +/- 1 ms vs. 8 +/- 1 ms; P < 0.05). 4. Neurons characterized as receiving a monosynaptic input from the aortic afferents generally did not exhibit time-dependent inhibition. Significant inhibition was observed only at a conditioning-test interval of 50 ms, when the average test response was 79 +/- 8% of control. In contrast, the average response following a 50-ms conditioning-test interval for neurons receiving polysynaptic input from the aortic nerve was only 32 +/- 8% of control. Significant inhibition was observed at conditioning-test intervals of up to 200 ms. 5. At a conditioning-test interval of 50 ms, only 5 of 22 monosynaptic neurons were inhibited by > 50%. Mean arterial pressure during the conditioning-test procedure was significantly lower for these neurons than for the 17 cells that were inhibited by < 50%. This suggests that the level of activity in convergent afferent input might influence the magnitude of time-dependent inhibition. 6. There was an essentially linear recovery from time-dependent inhibition evident in polysynaptic neurons that were tested at all conditioning-test intervals, suggesting a single mechanism of variable duration. Results reported here are consistent with current theory that time-dependent inhibition is mediated by disfacilitation. 7. The results demonstrate that NTS neurons receiving monosynaptic input from the aortic depressor nerve infrequently exhibit time-dependent inhibition. This could allow for the original, unmodified afferent information to be dispersed to subsequent neurons. In contrast, neurons receiving polysynaptic input undergo time-dependent inhibition similar to that which has been reported for other afferent inputs. This could allow for differential degrees of fidelity in the transfer of the afferent information to specific efferent pathways. Therefore the temporal pattern of firing in individual baroreceptor afferents could play a critical role in the function of the arterial baroreflex and therefore in the regulation of blood pressure.
1. We have developed a comprehensive mathematical model of an afferent synaptic connection to the soma of a medial nucleus tractus solitarius (mNTS) neuron. Model development is based on numerical fits to quantitative data recorded in our laboratory. This work is part of a continuing collaborative effort aimed at identifying and characterizing the mechanisms responsible for the non-linear integrative properties of this first synapse in the baroreceptor reflex. 2. The complete model consists of three major parts: 1) a Hodgkin-Huxley (HH)-type membrane model of the prejunctional sensory terminal bouton; 2) a multistage model describing vesicular storage, adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca(2+)-dependent mobilization, release and recycling; and 3) a HH-type membrane model of the postjunctional mNTS cell that includes descriptions for a desensitizing non-N-methyl-D-aspartate (NMDA) ionic current that is responsible for the fast excitatory postsynaptic potentials (EPSPs) observed in mNTS cells. The membrane models for both the terminal bouton and the mNTS neuron are coupled to separate lumped fluid compartment models describing intracellular Ca2+ ion concentration dynamics. 3. Our modeling strategy is twofold. The first is to validate model performance by reproducing a wide variety of experimental data both from our laboratory and from the literature. The second is to explore the functional aspects of the model in order to gain a greater appreciation for the balance between presynaptic mechanisms (e.g., terminal membrane properties and vesicular dynamics) and postsynaptic mechanisms (e.g., non-NMDA receptor kinetics and neuronal dynamics) that underlie the afferent synaptic drive of mNTS neurons. 4. The model accurately reproduces EPSP dynamics recorded with the use of a wide range of stimulus protocols. The model can also mirror the unique pattern of graded frequency- and use-dependent reduction in peak EPSP magnitude observed experimentally through 60 s of constant, suprathreshold synaptic activation. We demonstrate how vesicular mobilization, recycling, and receptor kinetics can function synergistically in establishing synaptic transfer. Furthermore, we show that by allowing the aggregate rate of vesicle mobilization to respond in a use-dependent manner, it is possible to compensate for the attenuating affects of desensitization at elevated rates of stimulation. 5. Our simulations indicate that the low-frequency characteristics of this synapse are dominated by vesicular dynamics, whereas the high-frequency properties arise from a combination of Ca(2+)-dependent vesicular mobilization and the kinetics of the non-NMDA receptor. Desensitization can influence the peak magnitude and decay time of the EPSP, thereby affecting synaptic throughput. However, we demonstrate that, as the time course of neurotransmitter in the synaptic cleft decreases, the influence of desensitization should be somewhat diminished. As a result, the effective bandwidth of the synapse increases and becomes limited by the gating characteristics of the non-NMDA channel. 6. The model also includes a neuromodulatory aspect in that the frequency response of the synapse can be modulated by an adenylate cyclase-mediated regulatory mechanism. Although our simulations indicate the behavior of a limited number of possible neuromodulatory agents, the results demonstrate the pivotal role such agents could play in modifying synaptic transfer characteristics presynaptically. 7. Both continuous and burst-mode tract stimulation evoke patterns of action potentials in spontaneously active mNTS neurons that are mimicked very well by our model. Our simulations demonstrate that, as the rate of stimulation increases beyond approximately 20-30 Hz, the inherent low-pass frequency-response characteristics of the synapse limit the overall dynamic range of the mNTS neuron, causing the postsynaptic cell to "entrain" at frequencies within its normal operating range.
1. Synaptic responses of medial nucleus tractus solitarius (mNTS) neurons to solitary tract (ST) activation were studied in a horizontal brain slice preparation of the rat medulla. Slices included sections of ST sufficiently long that the ST could be electrically activated several millimeters from the recording site of cell bodies in mNTS. 2. Three types of synaptic events were evoked in response to ST stimulation: simple excitatory postsynaptic potentials (EPSPs), simple inhibitory postsynaptic potentials (IPSPs), and complex EPSP-IPSP sequences. Simple EPSPs had substantially shorter latencies than IPSPs (3.39 +/- 0.65 ms, mean +/- SE, n = 42, vs. 5.86 +/- 0.71 ms, n = 6, respectively). 3. EPSP amplitude increased linearly with increasing hyperpolarization, with an extrapolated reversal potential near 0 mV. 4. EPSPs were maximal at < 0.5 Hz of sustained, constant-frequency ST stimulation (n = 14). EPSP amplitude declined to an average of 57.5% of control at 10 Hz after 2 s of sustained stimulation. With 1 min of sustained, 100-Hz stimulation, EPSP amplitude declined to near zero. 5. With stimuli intermittently delivered as 100-ms bursts every 300 ms, generally comparable average EPSPs were evoked during constant and burst patterns of ST stimulation. The amplitude of the initial EPSP in each burst was very well maintained even at intraburst stimulation rates of 100 Hz. 6. At resting membrane potentials, low constant frequencies of ST stimulation (< 5 Hz) reliably elicited action potentials and suppressed spontaneous spiking, but higher frequencies led to spike failures (> 85% at 100 Hz). Between 5 and 10 Hz, this periodic stimulation-suppression cycle clearly entrained action potential activity to the ST stimuli. Similar patterns of current pulses (5 ms) reliably evoked action potentials with each pulse to higher frequencies (50 Hz) without failures, and entrainment was similar to ST stimulation. 7. In a subset of nucleus tractus solitarius (NTS) neurons (3 of 9 studied), bursts of ST stimuli were as much as 50% more effective at transmitting high frequencies (> 10 Hz) of ST stimulation than the equivalent constant frequencies (P < 0.0001). 8. The long-latency simple IPSPs with no preceding EPSPs reversed to become depolarizing at potentials more negative than -62.9 +/- 7.0 mV (n = 5) and were blocked by the non-N-methyl-D-aspartate antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (n = 3). The ST stimulation frequency-response relation of these IPSPs was similar to that for the short-latency EPSP response excited by ST synapses. Thus these IPSPs appear to be activated polysynaptically via a glutamatergic-GABAergic sequence in response to ST activation. 9. The results suggest that sensory afferent synapses in mNTS have limited transmission of high-frequency inputs. Both synaptic transmission and the characteristics of the postsynaptic neuron importantly contribute to the action potential transmission from afferent to NTS neuron and beyond. This overall frequency response limitation may contribute to the accommodation of reflex responses from sensory afferent inputs such as arterial baroreceptors within their physiological discharge frequency range.
Neurons in the nucleus of the solitary tract (NTS) of the anesthetized rat were classified according to their responses to aortic depressor nerve stimulation: monosynaptic neurons (MSNs), polysynaptic neurons (PSNs) and non-aortic depressor nerve-evoked neurons (NENs). Agonists for excitatory amino acid (EAA) receptors were applied by microiontophoresis at currents of 5 to 40 nA. At these "doses," the nonselective EAA agonist glutamate (100 mM) increased the firing rate of some MSNs (5/9), PSNs (6/8) and NENs (16/20) (P < .01 for each group). Some neurons in each group were very resistant to glutamate, even at high ejecting currents. In addition, most NTS neurons were excited by selective EAA agonists, (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (10 mM), kainate (10 mM), N-methyl-D-aspartic acid (100 mM) and trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (100 mM). As with glutamate, some NTS neurons in each class were also very resistant to selective EAA agonists. Statistical analysis indicated that N-methyl-D-aspartic acid, but not (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate, was more potent on PSNs than on MSNs or NENs (P < .01 for each comparison). There was a trend for trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid to be more potent on MSNs than on PSNs or NENs (P = .09 and .07, respectively). Our results suggest that all EAA receptor subtypes are involved in baroreceptor afferent integration within NTS, and NTS neurons appear to possess different combinations of EAA receptor subtypes.
The central integration of signals from pulmonary vagal C-fibers (or type-J receptors) with those arising from cardiac, peripheral chemoreceptor, and baroreceptor afferents to neurons within the nucleus of the solitary tract (NTS) was studied in an arterially perfused working heart-brain stem preparation of adult mouse. Pulmonary vagal C-fibers were excited by right atrial injection of phenylbiguanide (PBG) while cardiac receptors were stimulated by left ventricular injection of veratridine (1-3 micrograms/kg) or mechanically by distension of the left ventricle (20-50 microl perfusate) using an indwelling cannula. Carotid body chemoreceptors were activated by aortic injection of Na cyanide, whereas baroreceptors were stimulated by increasing arterial perfusion pressure. Stimulation of pulmonary C-fibers and cardiac, chemo-, and baroreceptors all produced a reflex bradycardia (23-133 bpm). Central respiratory activity, as recorded from the phrenic nerve, was depressed by stimulating pulmonary C-fibers and cardiac and baroreceptors but enhanced in amplitude and frequency during chemoreceptor stimulation. Twenty-seven NTS neurons were excited and three were inhibited after pulmonary C-fiber stimulation displaying decrementing discharges with a peak firing frequency of up to 42 Hz (15 +/- 2.2 Hz, mean +/- SE) that lasted for 8.8 +/- 0.9 s. These responses occurred <1 s from the end of the PBG injection that was within the pulmonary circulation time. None of these cells responded to increases in right atrial pressure. All cells excited by PBG were also driven synaptically after electrical stimulation of the ipsilateral cervical vagus nerve at a latency of 32.9 +/- 3.2 ms (range 20-62 ms). None of these neurons had ongoing activity related to central respiratory activity. Convergence from cardiorespiratory afferents to 21 neurons driven by pulmonary C-fibers was tested. Twenty-five percent of cells were selectively excited by chemical stimulation of cardiac receptors alone, 19% were driven by peripheral chemoreceptors, and 38% responded to both cardiac and chemoreceptor activation. In contrast, only 13% of the cells activated by PBG injection responded to stimulation of baroreceptors and only 6% to cardiac mechanoreceptor stimulation. None of these neurons were activated by increasing right atrial pressure. The data indicate a high proportion of afferent convergence from pulmonary C-fibers, cardiac receptors, and peripheral chemoreceptors in the NTS. However, these neurons appear not to integrate inputs from cardiovascular mechanoreceptors. The significance of the data is discussed in relation to pathological disease states such as pulmonary congestion and cardiac failure.
In an earlier study, we examined the pressure-response characteristics of rat aortic baroreceptors with C-fibre (non-medullated) afferents. Compared with aortic baroreceptor fibres with A-fibre (medullated) afferents, the C-fibres were activated at higher pressures and discharged more irregularly when stimulated with a steady level of pressure. Here we examine the relationship between discharge and the aortic diameter in these two types of afferents in rats and rabbits. An in vitro aortic arch/aortic nerve preparation was used to record single-fibre activity simultaneously with aortic arch pressure and diameter. Diameter was measured using a highly sensitive non-contact photoelectric device. Baroreceptor discharge was characterized by stimulating the nerve endings with either slow pressure ramps from subthreshold to 200-250 mmHg, at a rate of rise of 2 mmHg s-1, or pressure steps from subthreshold to suprathreshold levels, at amplitudes of 110-180 mmHg. In response to these inputs, C-fibres in rabbits (conduction velocities= 0.8-2.2 m s-1) behaved much like those in rats. The C-fibres had significantly higher pressure thresholds (95 +/- 3 mmHg vs. 53 +/- 2 mmHg; mean +/- SEM), lower threshold frequencies (2.4 +/- 0.5 vs. 27.7 +/- 1.8 spikes s-1), lower maximum discharge frequencies (22.7 +/- 2.3 vs. 65 +/- 5.8 spikes s-1) and more irregular discharge in response to a pressure step when compared with A-fibres (conduction velocities of 8-16 m s-1). When plotted against diameter, C-fibre ramp-evoked discharge increased gradually at first, and then rose steeply at increasingly higher ramp pressures where aortic diameter became relatively constant. In contrast, A-fibre discharge was linearly related to diameter over a wide range of pressure. These results suggest two interpretations: (1) The relation between stretch and C-fibre discharge is highly non-linear, with a marked increase in sensitivity at large diameters. (2) C-fibres are stimulated by changes in intramural stress rather than stretch.
1. Vagal afferent input from cardiac mechanoreceptors excites neurones in the nucleus tractus solitarii (NTS), but discharge patterns evoked by physiological activation of pressure-sensitive cardiac mechanoreceptors have not been studied in vivo. The role of glutamate receptor subtypes in transmission of afferent activity to the NTS neurones has not been determined. The present study therefore has two aims: first, to characterise the discharge patterns of neurones in the NTS that receive pressure-sensitive vagal cardiac receptor input and second, to determine the roles of ionotropic glutamate receptor subtypes in the transmission of this putative cardiac mechanoreceptor-related activity to NTS neurones. 2. Pulse-synchronous activity of neurones in the NTS evoked by vagal afferent input was recorded extracellularly in an anaesthetised dog model using multibarrel glass electrodes, which allowed picoejection of the glutamate receptor antagonists NBQX or AP5 to block either non-NMDA or NMDA receptors, respectively, during the neuronal recording. Pressure sensitivity of the recorded neurones was examined by monitoring their response to a small increase in arterial blood pressure. Selective pressure activation of carotid sinus baroreceptors in an isolated sinus or selective denervation of aortic baroreceptors were used to test for convergent excitation of the neurones by arterial baroreceptors. 3. Pulse-synchronous cardiac-related neuronal activity recorded from neurones in both the right and left NTS was eliminated following section of the left (n = 17) or right (n = 1) vagus nerves. No spontaneous, non-pulsatile activity was observed in these neurones before or after vagotomy. Activity transmitted via left vagal afferents was found to be sensitive to changes in arterial blood pressure. In these neurones, activity was blocked in 13 of 17 neurones by picoejection of NBQX, with the remainder requiring both NBQX and AP5. None of the cardiac-related neurones responded to activation of carotid baroreceptors or denervation of aortic baroreceptors, indicating no convergence of activity from carotid baroreceptors or aortic baroreceptors with pressure thresholds of approximately 130 mmHg or less. 4. The results suggest that vagal pressure-sensitive afferent input from cardiac mechanoreceptors is transmitted primarily by left vagal afferent fibres via non-NMDA receptors to neurones in both the ipsilateral and contralateral NTS. NMDA receptors were also found to have a role in the activation of a small subpopulation of neurones.
The increased release of oxytocin during lactation has been shown to be dependent upon glutamatergic transmission and is associated with an increased synaptic innervation of the supraoptic nucleus (SON). To determine whether the glutamatergic synaptic properties of oxytocin neurones are changed during lactation, we recorded excitatory postsynaptic currents (EPSCs) from identified oxytocin neurones in the SON of slices taken from adult virgin and lactating rats. The frequency of AMPA-mediated miniature EPSCs (mEPSCs) more than doubled during lactation. In addition, the decay time constant, but not the amplitude of the mEPSCs was significantly increased in both vasopressin and oxytocin neurones. Paired-pulse facilitation (PPF) was significantly reduced in oxytocin neurones during lactation, whereas no change was observed in vasopressin neurones. Elevating Ca(2+) reduced PPF in oxytocin neurones in virgin rats but did not alter PPF in oxytocin neurones from lactating rats. Collectively, our results suggest that excitatory glutamatergic transmission is strengthened in oxytocin neurones during lactation, probably by a combination of an increased number of terminals, slower decay kinetics, and an increase in the probability of release.