Cholinergic collaterals arising from noradrenergic sympathetic neurons in mice.

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. 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.

Brainstem astrocytes control homeostatic regulation of caloric intake.

Prolonged high-fat diet (HFD) exposure is associated with hyperphagia, excess caloric intake and weight gain. After initial exposure to a HFD, a brief (24-48 h) period of hyperphagia is followed by the regulation of caloric intake and restoration of energy balance within an acute (3-5 day) period. Previous studies have demonstrated this occurs via a vagally mediated signalling cascade that increases glutamatergic transmission via activation of NMDA receptors located on gastric-projecting neurons of the dorsal motor nucleus of the vagus (DMV). The present study used electrophysiological recordings from thin brainstem slice preparations, in vivo recordings of gastric motility and tone, measurement of gastric emptying rates, and food intake studies to investigate the hypothesis that activation of brainstem astrocytes in response to acute HFD exposure is responsible for the increased glutamatergic drive to DMV neurons and the restoration of caloric balance. Pharmacological and chemogenetic inhibition of brainstem astrocytes reduced glutamatergic signalling and DMV excitability, dysregulated gastric tone and motility, attenuated the homeostatic delay in gastric emptying, and prevented the decrease in food intake that is observed during the period of energy regulation following initial exposure to HFD. Understanding the mechanisms involved in caloric regulation may provide critical insights into energy balance as well as into the hyperphagia that develops as these mechanisms are overcome. KEY POINTS: Initial exposure to a high fat diet is associated with a brief period of hyperphagia before caloric intake and energy balance is restored. This period of homeostatic regulation is associated with a vagally mediated signalling cascade that increases glutamatergic transmission to dorsal motor nucleus of the vagus (DMV) neurons via activation of synaptic NMDA receptors. The present study demonstrates that pharmacological and chemogenetic inhibition of brainstem astrocytes reduced glutamatergic signalling and DMV neuronal excitability, dysregulated gastric motility and tone and emptying, and prevented the regulation of food intake following high-fat diet exposure. Astrocyte regulation of glutamatergic transmission to DMV neurons appears to involve release of the gliotransmitters glutamate and ATP. Understanding the mechanisms involved in caloric regulation may provide critical insights into energy balance as well as into the hyperphagia that develops as these mechanisms are overcome.

Untangling Peripheral Sympathetic Neurocircuits.

The sympathetic nervous system plays a critical role in regulating many autonomic functions, including cardiac rhythm. The postganglionic neurons in the sympathetic chain ganglia are essential components that relay sympathetic signals to target tissues and disruption of their activity leads to poor health outcomes. Despite this importance, the neurocircuitry within sympathetic ganglia is poorly understood. Canonically, postganglionic sympathetic neurons are thought to simply be activated by monosynaptic inputs from preganglionic cholinergic neurons of the intermediolateral cell columns of the spinal cord. Early electrophysiological studies of sympathetic ganglia where the peripheral nerve trunks were electrically stimulated identified excitatory cholinergic synaptic events in addition to retrograde action potentials, leading some to speculate that excitatory collateral projections are present. However, this seemed unlikely since sympathetic postganglionic neurons were known to synthesize and release norepinephrine and expression of dual neurochemical phenotypes had not been well recognized. In vitro studies clearly established the capacity of cultured sympathetic neurons to express and release acetylcholine and norepinephrine throughout development and even in pathophysiological conditions. Given this insight, we believe that the canonical view of ganglionic transmission needs to be reevaluated and may provide a mechanistic understanding of autonomic imbalance in disease. Further studies likely will require genetic models manipulating neurochemical phenotypes within sympathetic ganglia to resolve the function of cholinergic collateral projections between postganglionic neurons. In this perspective article, we will discuss the evidence for collateral projections in sympathetic ganglia, determine if current laboratory techniques could address these questions, and discuss potential obstacles and caveats.

What gets on the nerves of cardiac patients? Pathophysiological changes in cardiac innervation.

The autonomic nervous system regulates cardiac function by balancing the actions of sympathetic and parasympathetic inputs to the heart. Intrinsic cardiac neurocircuits integrate these autonomic signals to fine-tune cardiac control, and sensory feedback loops regulate autonomic transmission in the face of external stimuli. These interconnected neural systems allow the heart to adapt to constantly changing circumstances that range from simple fluctuations in body position to running a marathon. The cardiac reflexes that serve to maintain homeostasis in health are disrupted in many disease states. This is often characterized by increased sympathetic and decreased parasympathetic transmission. Studies of cardiovascular disease reveal remodelling of cardiac neurocircuits at several functional and anatomical levels. Central circuits change so that sympathetic pathways become hyperactive, while parasympathetic circuits exhibit decreased activity. Peripheral sensory nerves also become hyperactive in disease, which increases patients' risk for poor cardiac outcomes. Injury and disease also alter the types of neurotransmitters and neuropeptides released by autonomic nerves in the heart, and can lead to regional hyperinnervation (increased nerve density) or denervation (decreased nerve density) of cardiac tissue. The mechanisms responsible for neural remodelling are not fully understood, but neurotrophins and inflammatory cytokines are likely involved. Areas of active investigation include the role of immune cells and inflammation in neural remodelling, as well as the role of glia in modulating peripheral neuronal activity. Our growing understanding of autonomic dysfunction in disease has facilitated development of new therapeutic strategies to improve health outcomes.

DMV extrasynaptic NMDA receptors regulate caloric intake in rats.

Acute high-fat diet (aHFD) exposure induces a brief period of hyperphagia before caloric balance is restored. Previous studies have demonstrated that this period of regulation is associated with activation of synaptic N-methyl-D-aspartate (NMDA) receptors on dorsal motor nucleus of the vagus (DMV) neurons, which increases vagal control of gastric functions. Our aim was to test the hypothesis that activation of DMV synaptic NMDA receptors occurs subsequent to activation of extrasynaptic NMDA receptors. Sprague-Dawley rats were fed a control or high-fat diet for 3-5 days prior to experimentation. Whole-cell patch-clamp recordings from gastric-projecting DMV neurons; in vivo recordings of gastric motility, tone, compliance, and emptying; and food intake studies were used to assess the effects of NMDA receptor antagonism on caloric regulation. After aHFD exposure, inhibition of extrasynaptic NMDA receptors prevented the synaptic NMDA receptor-mediated increase in glutamatergic transmission to DMV neurons, as well as the increase in gastric tone and motility, while chronic extrasynaptic NMDA receptor inhibition attenuated the regulation of caloric intake. After aHFD exposure, the regulation of food intake involved synaptic NMDA receptor-mediated currents, which occurred in response to extrasynaptic NMDA receptor activation. Understanding these events may provide a mechanistic basis for hyperphagia and may identify novel therapeutic targets for the treatment of obesity.

Glutamatergic plasticity within neurocircuits of the dorsal vagal complex and the regulation of gastric functions.

The meticulous regulation of the gastrointestinal (GI) tract is required for the coordination of gastric motility and emptying, intestinal secretion, absorption, and transit as well as for the overarching management of food intake and energy homeostasis. Disruption of GI functions is associated with the development of severe GI disorders and the alteration of food intake and caloric balance. Functional GI disorders as well as the dysregulation of energy balance and food intake are frequently associated with, or result from, alterations in the central regulation of GI control. The faithful and rapid transmission of information from the stomach and upper GI tract to second-order neurons of the nucleus of the tractus solitarius (NTS) relies on the delicate modulation of excitatory glutamatergic transmission, as does the relay of integrated signals from the NTS to parasympathetic efferent neurons of the dorsal motor nucleus of the vagus (DMV). Many studies have focused on understanding the physiological and pathophysiological modulation of these glutamatergic synapses, although their role in the control and regulation of GI functions has lagged behind that of cardiovascular and respiratory functions. The purpose of this review is to examine the current literature exploring the role of glutamatergic transmission in the DVC in the regulation of GI functions.

Perinatal high-fat diet alters development of GABAA receptor subunits in dorsal motor nucleus of vagus.

Perinatal high-fat diet (pHFD) exposure increases the inhibition of dorsal motor nucleus of the vagus (DMV) neurons, potentially contributing to the dysregulation of gastric functions. The aim of this study was to test the hypothesis that pHFD increases the inhibition of DMV neurons by disrupting GABAA receptor subunit development. In vivo gastric recordings were made from adult anesthetized Sprague-Dawley rats fed a control or pHFD (14 or 60% kcal from fat, respectively) from embryonic day 13 (E13) to postnatal day 42 (P42), and response to brainstem microinjection of benzodiazepines was assessed. Whole cell patch clamp recordings from DMV neurons assessed the functional expression of GABAA α subunits, whereas mRNA and protein expression were measured via qPCR and Western blotting, respectively. pHFD decreased basal antrum and corpus motility, whereas brainstem microinjection of L838,417 (positive allosteric modulator of α2/3 subunit-containing GABAA receptors) produced a larger decrease in gastric tone and motility. GABAergic miniature inhibitory postsynaptic currents in pHFD DMV neurons were responsive to L838,417 throughout development, unlike control DMV neurons, which were responsive only at early postnatal timepoints. Brainstem mRNA and protein expression of the GABAA α1,2, and 3 subunits, however, did not differ between control and pHFD rats. This study suggests that pHFD exposure arrests the development of synaptic GABAA α2/3 receptor subunits on DMV neurons and that functional synaptic expression is maintained into adulthood, although cellular localization may differ. The tonic activation of slower GABAA α2/3 subunit-containing receptors implies that such developmental changes may contribute to the observed decreased gastric motility. NEW & NOTEWORTHY Vagal neurocircuits involved in the control of gastric functions, satiation, and food intake are subject to significant developmental regulation postnatally, with immature GABAA receptors expressing slower α2/3-subunits, whereas mature GABAA receptor express faster α1-subunits. After perinatal high-fat diet exposure, this developmental regulation of dorsal motor nucleus of the vagus (DMV) neurons is disrupted, increasing their tonic GABAergic inhibition, decreasing efferent output, and potentially decreasing gastric motility.

Role of astroglia in diet-induced central neuroplasticity.

Obesity, characterized by increased adiposity that develops when energy intake outweighs expenditure, is rapidly becoming a serious health crisis that affects millions of people worldwide and is associated with severe comorbid disorders including hypertension, cardiovascular disease, and type II diabetes. Obesity is also associated with the dysregulation of central neurocircuits involved in the control of autonomic, metabolic, and cognitive functions. Systemic inflammation associated with diet-induced obesity (DIO) has been proposed to be responsible for the development of these comorbidities as well as the dysregulation of central neurocircuits. A growing body of evidence suggests, however, that exposure to a high-fat diet (HFD) may cause neuroinflammation and astroglial activation even before systemic inflammation develops, which may be sufficient to cause dysregulation of central neurocircuits involved in energy homeostasis before the development of obesity. The purpose of this review is to summarize the current literature exploring astroglial-dependent modulation of central circuits following exposure to HFD and DIO, including not only dysregulation of neurocircuits involved in energy homeostasis and feeding behavior, but also the dysregulation of learning, memory, mood, and reward pathways.

High-Fat Diet During the Perinatal Period Induces Loss of Myenteric Nitrergic Neurons and Increases Enteric Glial Density, Prior to the Development of Obesity.

Diet-induced obesity induces peripheral inflammation accompanied by a loss of myenteric neurons. Few studies, however, have investigated the effects of a high-fat diet (HFD) on either the development of myenteric neurons or prior to the occurrence of obesity. The present study assessed the effects of maternal HFD on the density and neurochemical phenotype of myenteric ganglia in the upper gastrointestinal tract. Sprague-Dawley rats were fed either a control or HFD (14% or 60% kcal from fat, respectively) from embryonic day 13; the fundus, corpus and duodenum were fixed thereafter at postnatal 2, 4, 6 and 12 weeks of age for subsequent immunohistochemical studies. While myenteric ganglion size did not differ throughout the study, HFD exposure decreased the number of nitrergic neurons by 6 weeks of age in all regions. This decrease was accompanied by a loss of PGP-immunoreactive neurons, suggesting a decline in myenteric neuronal number. HFD also increased myenteric plexus glial cell density in all regions by 4 weeks of age. These changes occurred in the absence of an increase in serum or gastric inflammatory markers. The present study suggests that exposure to a HFD during the perinatal time period results in glial proliferation and loss of inhibitory nitrergic neurons prior to the onset of obesity, suggesting that dietary alterations may affect gastrointestinal functions independently of increased adiposity or glycemic dysregulation.

Acute high-fat diet upregulates glutamatergic signaling in the dorsal motor nucleus of the vagus.

Obesity is associated with dysregulation of vagal neurocircuits controlling gastric functions, including food intake and energy balance. In the short term, however, caloric intake is regulated homeostatically although the precise mechanisms responsible are unknown. The present study examined the effects of acute high-fat diet (HFD) on glutamatergic neurotransmission within central vagal neurocircuits and its effects on gastric motility. Sprague-Dawley rats were fed a control or HFD diet (14% or 60% kcal from fat, respectively) for 3-5 days. Whole cell patch-clamp recordings and brainstem application of antagonists were used to assess the effects of acute HFD on glutamatergic transmission to dorsal motor nucleus of the vagus (DMV) neurons and subsequent alterations in gastric tone and motility. After becoming hyperphagic initially, caloric balance was restored after 3 days following HFD exposure. In control rats, the non- N-methyl-d-aspartate (NMDA) receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX), but not the NMDA receptor antagonist, amino-5-phosphonopentanoate (AP5), significantly decreased excitatory synaptic currents and action potential firing rate in gastric-projecting DMV neurons. In contrast, both AP5 and DNQX decreased excitatory synaptic transmission and action potential firing in acute HFD neurons. When microinjected into the brainstem, AP5, but not DNQX, decreased gastric motility and tone in acute HFD rats only. These results suggest that acute HFD upregulates NMDA receptor-mediated currents, increasing DMV neuronal excitability and activating the vagal efferent cholinergic pathway, thus increasing gastric tone and motility. Although such neuroplasticity may be a persistent adaptation to the initial exposure to HFD, it may also be an important mechanism in homeostatic regulation of energy balance. NEW & NOTEWORTHY Vagal neurocircuits are critical to the regulation of gastric functions, including satiation and food intake. Acute high-fat diet upregulates glutamatergic signaling within central vagal neurocircuits via activation of N-methyl-d-aspartate receptors, increasing vagal efferent drive to the stomach. Although it is possible that such neuroplasticity is a persistent adaptation to initial exposure to the high-fat diet, it may also play a role in the homeostatic control of feeding.