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Coordinated autonomic and respiratory responses evoked by alerting stimuli: Role of the midbrain colliculi
Respiratory Physiology & Neurobiology 226 (2016) 87–93
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Coordinated autonomic and respiratory responses evoked by alerting stimuli: Role of the midbrain colliculi Flávia C.F. Müller-Ribeiro a,b,∗ , Ann K. Goodchild a , Simon McMullan a , Marco A.P. Fontes b , Roger A.L. Dampney c a
Australian School of Advanced Medicine, Macquarie University, NSW 2109, Australia Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais (UFMG), Minas Gerais 31270-901, Brazil c School of Medical Sciences (Physiology) and Bosch Institute, University of Sydney, NSW 2006, Australia b
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Article history: Received 7 August 2015 Received in revised form 21 October 2015 Accepted 23 October 2015 Available online 10 November 2015 Keywords: Superior colliculus Inferior colliculus Alerting response Respiratory activity Respiratory–sympathetic coupling Command neuron
a b s t r a c t Threatening stimuli trigger rapid and coordinated behavioral responses supported by cardiorespiratory changes. The midbrain colliculi can generate coordinated orienting or defensive behavioral responses, and it has been proposed that collicular neurons also generate appropriate cardiovascular and respiratory responses to support such behaviors. We have shown previously that under conditions where collicular neurons are disinhibited, coordinated cardiovascular, somatomotor and respiratory responses can be evoked independently of the cortex by auditory, visual and somatosensory stimuli. Here we report that these natural stimuli effectively increase inspiratory time most likely though phase switching. As a result the pattern of phrenic and sympathetic coupling is an inspiratory-related sympathoexcitation. We propose that blockade of tonic GABAergic input in the midbrain colliculi permits alerting stimuli to drive command neurons that generate coordinated cardiovascular, respiratory and motor outputs. The outputs of these command neurons likely interact with the central respiratory pattern generator, however the precise output pathways mediating the coordinated autonomic and respiratory responses remain to be determined. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The acute alerting response to threatening stimuli consists of an integrated and stereotyped pattern of autonomic, respiratory and somatomotor changes that increases the probability of survival and is found in all species. Interestingly, the physiological responses seen in animals exposed to diverse environmental challenges include specific stereotyped changes. A sudden alerting stimulus, usually signaled by a visual, auditory or somatosensory input, immediately generates a behavioral response such as avoidance or flight depending on the intensity of the stimulus (Dampney et al., 2008; Kabir et al., 2010). The behavioral response is always accompanied by autonomic changes characterized by increases in blood pressure (BP), tachycardia, mydriasis, piloerection and cutaneous vasoconstriction (Hilton, 1982; Dampney et al., 2008). Respiratory changes of rapid onset, which consist of increases in respiratory rate and depth, are also observed in an alerting response
∗ Corresponding author at: Instituto de Ciências Biológicas, Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, Minas Gerais, Brazil. E-mail address: fl[email protected] (F.C.F. Müller-Ribeiro). http://dx.doi.org/10.1016/j.resp.2015.10.012 1569-9048/© 2015 Elsevier B.V. All rights reserved.
(Blanchard and Blanchard, 1972; Kabir et al., 2010). The increase in arterial pressure provides perfusion of skeletal muscles priming them for activity, whereas, the increase in respiratory activity optimizes gas exchange to match increased activity or vigilance optimizing the fight/flight reaction (Dampney et al., 2008). Previous studies have indicated that sites within the central nervous system such as the dorsomedial hypothalamus, periaqueductal grey (PAG), amygdala and the midbrain colliculi are involved in the generation of coordinated aversive responses to threatening stimuli (Dampney et al., 2013; Dampney, 2015). However, the pattern of the evoked responses vary greatly with the nature of the danger; depending upon whether the threat is present and requires immediate action, or simply enhanced and sustained vigilance (Blanchard et al., 1993). 2. Role of the midbrain colliculi in the alerting response The superior colliculus (SC) is a layered region localized in the dorsal region of the midbrain. The superficial layers receive a direct innervation from the retina and an indirect one from the visual cortex, whereas the intermediate and deep layers receive auditory and somatosensory inputs (Dean et al., 1989). Electrical and chemical
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Fig. 1. Examples of recordings from two different experiments: intact brain and following decerebration. (A) Cardiovascular, respiratory and somatomotor responses evoked by pinch after microinjection of picrotoxin into the colliculus. (B) Cardiovascular, sympathetic and respiratory responses evoked by another sensory stimulus (touching whiskers) after microinjection of picrotoxin into the colliculus. (C) Cardiovascular and respiratory responses evoked by pinch after microinjection of bicuculline into the colliculus in a decerebrate preparation.
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Fig. 2. Schematic diagram showing the subcortical loop involving the superior colliculus (SC) and basal ganglia, based on a scheme proposed by McHaffie et al. (2005). The deep layers of the SC receive topographically organized visual, auditory, and somatosensory inputs, whereas, superficial layers receive only visual inputs. There are multiple parallel projections from the deep layers of the SC to the striatum, relayed via intralaminar nuclei (ILN) in the thalamus. The striatum also receives inputs from the cortex, amygdala and possibly also the dorsomedial hypothalamus (DMH), and may act as an action selector, comparing inputs from the SC with those from the cortex, amygdala and DMH. For further details see the text. The red lines indicate an excitatory projection, whereas, the blue lines indicate an inhibitory projection. Modified from Fig. 7 in Ref. Dampney (2015). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Fig. 3. (A) Example of auditory-evoked responses with SpSNA, PNA and VNA burst synchrony after microinjection of picrotoxin into the midbrain colliculi. The vertical shaded line indicates the blockade of the post inspiratory peak by the auditory stimulus. (B) Portion of the single clap response showing phrenic nerve activity overlaying vagus nerve activity in a normal breathing cycle and after auditory stimulus. SpSNA: splanchnic nerve activity, PNA: phrenic nerve activity, VNA: vagus nerve activity, I: inspiration, PI: postinspiration (stage 1 expiration), LE: late expiration (stage 2 expiration), SNA: sympathetic nerve activity.
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Fig. 4. Examples of different respiratory-sympathetic coupling patterns. (A) Respiratory-sympathetic response under resting conditions in which the peak of sympathetic activity occurs during the PI phase. (B) Phrenic-triggered averages in sympathetic nerve activity during hypoxia in which the peak of sympathetic nerve activity occurs during the LE phase. (C) Averaged sympathetic and respiratory responses to alerting stimuli following collicular disinhibition showing that increases in sympathetic activity exhibited peaks of discharge coincident with inspiratory discharge. The traces in A and B are re-drawn from Figs. 1C and 5B in Ref. Dick et al. (2004), with permission. Abbreviations: SNA: sympathetic nerve activity, PNA: phrenic nerve activity.
stimulation of the SC suggest that it is involved in the generation of two types of behavioral responses to novel sensory stimuli: an orienting response toward a stimulus or a defensive response such as avoidance or flight (Dean et al., 1989). It appears that the type of the behavioral response generated depends on the degree of threat. Lesions of the SC reduce behavioral responses to visual stimuli and indicate a major role in defensive responses (Goodale and Murison, 1975). An appropriate cardiorespiratory response would be expected to match this behavioral response as part of a coordinated response to the appearance or sound of a predator, for example. Consistent with this, increases in blood pressure, heart rate as well as respiratory rate and depth were observed following stimulation of neurons within the SC (Keay et al., 1988). The pattern of these cardiorespiratory responses varied depending on the injection site, with more pronounced responses obtained after stimulation of the intermediate and deep layers. The inferior colliculus (IC) is a well known relay station for auditory pathways in the midbrain. Similar to the SC, functional evidence supports a role in the generation of defensive responses. In particular, electrical or chemical stimulation of this region elicits behaviors characterized by alertness, freezing or escape (Brandao et al., 1993) and again the responses were accompanied by autonomic changes typically seen when the animal is confronted with a threatening stimuli (Brandao et al., 1993). Together, these studies suggest that the midbrain collicular region is capable of generating integrated behavioral and autonomic responses when appropriately triggered. In accordance with this idea, we have previously shown that disinhibition of neurons localized within the intermediate and deep layers of the superior colliculus and the central and external nuclei of the inferior colliculus in anesthetized rats, resulted in a response characterized by highly synchronized sympathetic and respiratory bursts of nerve activity, as well as increases in blood pressure and heart rate that resemble the autonomic and respiratory response triggered by alerting stimuli (Iigaya et al., 2012). If this region is involved in the generation of a coordinated cardiorespiratory response and receives auditory, visual and somatosensory inputs, it would be expected that an alerting stimulus could evoke the same pattern of response. Recently, we tested this hypothesis and showed that in anesthetized rats an auditory (sound of a clap), visual (flash of light) or somatosensory (pinch) stimulus generated immediate and
coordinated sympathetic, respiratory and somatomotor changes followed by increases in blood pressure, but only after disinhibition of neurons within the collicular region (Fig. 1A) (Müller-Ribeiro et al., 2014). In 8 experiments, a non-nociceptive somatosensory stimulus (touching whiskers) was used which also generated the same pattern of coordinated response (Fig. 1B). The responses were highly synchronized, with short intervals (20–50 ms) between the onsets of the somatomotor, respiratory and sympathetic responses with the onset of the somatomotor burst preceding the onset of the respiratory and then the sympathetic bursts. If a common population of neurons within the colliculi generates these responses, such differences in onset latencies would be expected because postganglionic sympathetic nerve fibers are unmyelinated and have a slower conduction velocity than myelinated axons of phrenic and sciatic motoneurons (Iigaya et al., 2012; Müller-Ribeiro et al., 2014). These observations led us to propose that these responses are driven by a common population of command neurons localized within the SC and IC (Müller-Ribeiro et al., 2014). Remarkably, auditory and somatosensory-evoked responses were still intact after removal of the entire forebrain (Fig. 1C). Therefore, the essential circuitry subserving these responses under conditions in which GABAergic inhibition to the collicular region is blocked in anesthetized rats, does not include the amygdala, hypothalamus, PAG, and prefrontal cortex, all regions known to mediate cardiovascular, respiratory and behavioral responses to stressful situations. At the same time, previous studies in conscious rats have shown that inhibition of the amygdala or the dorsomedial hypothalamus greatly reduces the increases in respiratory rate evoked by either a brief alerting visual or auditory stimulus or a more prolonged stressful stimulus (Bondarenko et al., 2014, 2015). A possible explanation that would reconcile our previous findings in anesthetized rats with those of Bondarenko et al. (2014, 2015) has been proposed by Dampney (2015) (Fig. 2), based on a scheme proposed by McHaffie et al. (2005). The substantia nigra pars reticulata (SNpr) is a major source of direct tonic GABAergic input to the colliculi (Redgrave et al., 1992; Castellan-Baldan et al., 2006). The SNpr itself receives inhibitory inputs from the striatum that in turn receives inputs from the deep layers of the SC relayed via the thalamus as well as inputs from the cortex and other forebrain regions (Castellan-Baldan et al., 2006; Groenewegen et al., 1999; McHaffie et al., 2005). The SNpr and striatum are components of
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the basal ganglia. The inputs to the SC from visual, auditory, and somatosensory inputs and their connections within this subcortical loop are highly topographically organized, with the result that the pattern of inputs to the striatum are highly specific for a particular stimulus (Dean et al., 1989; McHaffie et al., 2005). McHaffie et al. (2005) proposed that the striatum may act as an “action selector,” comparing inputs from the SC with those from the cortex, so that the most appropriate action can be selected for a particular set of inputs, by withdrawing inhibition from the particular output neurons in the SC that regulate the appropriate coordinated somatomotor/autonomic response. It is conceivable that direct or indirect descending pathways from the amygdala and dorsomedial hypothalamus also project to the striatum, and may be essential for the full expression of coordinated somatomotor, autonomic and respiratory changes generated by collicular neurons in response to alerting stimuli. In that case, inhibition of either the amygdala and dorsomedial hypothalamus would attenuate responses to alerting stimuli, as demonstrated by Bondarenko et al. (2014, 2015). It should also be noted that alerting stimuli in conscious rats can elicit extreme tachypnea or sniffing, with respiratory rates increasing up to 450–500 breaths/min (Bondarenko et al., 2015). Such extreme responses do not occur in anesthetized rats in response to stimulation of sites in the colliculi (Iigaya et al., 2012). This may be due to the depressant effect of anesthesia on the respiratory rate generator in the brainstem, or alternatively may reflect the fact, as stated above, that the full expression of the respiratory response to alerting stimuli depends upon structures in the forebrain as well as the colliculi. What output pathways from the colliculi could mediate the autonomic, respiratory and behavioral responses? The output pathways from the SC have been studied primarily with regard to motor function. Projections from the midbrain colliculi include a projection to the dorsolateral periaqueductal grey (PAG) (Rhoades et al., 1989), which regulates sympathetic and respiratory activity probably via ascending projections to the dorsomedial hypothalamus (de Menezes et al., 2009; Horiuchi et al., 2009). Although PAG and SC share some anatomical and functional properties, the circuitry mediating these responses seems to be different. There are also descending projections from the SC to the pontomedullary reticular nuclei, but these do not appear to target the rostral ventrolateral medulla or the dorsal and ventral respiratory groups, regions known to control sympathetic and respiratory activity (Redgrave et al., 1987). Similar, the neural pathways of the IC involved in generating the sympathetic, respiratory and somatomotor responses are still poorly understood. There is also a direct projection from the IC to the SC (Garcia del Cano et al., 2006) and it is conceivable that this connection may target neurons in the SC that evoke sympathetic, respiratory and somatomotor response. It is unclear, however, how the autonomic and respiratory output is processed and how this output is coordinated with the motor output.
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the PI or LE phase, the expiratory period was shortened, however, if the stimulus arrived during inspiration the duration of the inspiratory activity was either unchanged or increased but with little effect on the duration of the expiratory period (e.g., Fig. 7A in Ref. Müller-Ribeiro et al., 2014). In one experiment in that study, we also measured the vagal nerve activity (VNA) in addition to PNA, to observe more directly the effects on I, PI and LE. Fig. 3 shows examples of the respiratory responses evoked by auditory stimuli (after blockade of GABA receptors in the colliculi) in this experiment. Each stimulus selectively blocked the post inspiratory peak in the vagus nerve (VNA) effectively shortening the subsequent expiratory period as can be seen in the recordings of PNA and VNA (Fig. 3). In this experiment, there were 103 respiratory cycles in which the pattern of VNA activity was altered following a stimulus, and in which the 3 phases of the respiratory cycle could be identified from the VNA recording. Out of these 103 cycles, the PI peak was abolished in 58 cases (56%) and decreased in the remaining 45 cycles (44% of the total). In contrast, the stimulus had little effect on the I peak (unchanged in 87 cases, increased in 10 cases and decreased in 6 cases). Statistical analysis revealed that these different effects on the PI and I peak were highly statistically significant (Chi-squared test, P < 0.001). Such a selective effect on the PI component of the respiratory cycle has only been generated from three regions, all located in the brainstem: the Kölliker-fuse nucleus, Bötzinger complex and the nucleus tractus solitarius (NTS) (Dutschmann et al., 2014). Inhibition or removal of the Kölliker-fuse nucleus evokes a similar pattern of activity in the vagus nerve (Smith et al., 2007; Bautista and Dutschmann, 2014; Dutschmann and Herbert, 2006). Injection of the inhibitory peptide somatostatin in the Bötzinger complex selectively abolishes post inspiratory activity (Burke et al., 2010) whereas, blockade of glutamate receptors in the (NTS) reduces postinspiratory activity (Costa-Silva et al., 2010). It is therefore possible that one or more of these candidate regions may receive inputs from the midbrain colliculi which may modify timing or phase switching of the respiratory cycle. However, the respiratory effect following sensory stimuli after disinhibition of the colliculus was dependent upon the phase of the respiratory cycle in which the stimulus arrived. The inhibition of postinspiratory activity occurred when the stimulus arrived during LE. If the stimulus arrived during PI the expiratory period immediately following was also shortened (Fig. 1C) however, when the stimulus arrived during inspiration the inspiratory activity was lengthened and there was little effect on the expiratory period (Fig. 1A). Additional examples of this phase switching are also evident in our previously published work (Müller-Ribeiro et al., 2014, Fig. 7). All of these patterns effectively increase the period of inspiration when the animal receives an alerting stimulus. These data strongly indicate that the output pathways activated by the stimuli must interact with the central pattern generator. The inhibition of the PI component decreases the expiratory period and thus increases respiratory frequency when the animal receives an alerting stimulus.
3. Effects on the respiratory pattern generator of natural stimuli mediated by the colliculi The central pattern generator comprising premotor respiratory neurons in pontomedullary locations (Bautista and Dutschmann, 2014) produces a breathing cycle composed of 3 phases: inspiration (I), post inspiration (PI) and late expiration (LE) (Fig. 3A), which can be identified in the integrated activity of phrenic and vagus nerves (Richter, 1996; Richter and Spyer, 2001). In our previously published study (Müller-Ribeiro et al., 2014), we observed that when stimuli evoked respiratory responses following collicular disinhibition, the effect on the pattern of phrenic activity (PNA) depended on the timing of the stimulus with respect to the phase of the respiratory cycle. In particular, if the stimulus arrived during
4. Pattern of respiratory–sympathetic coupling generated by inputs from the colliculi Sympathetic nerve activity is modulated by the respiratory system via connections from respiratory neurons in the lower brainstem to sympathetic pre motor neurons (Haselton and Guyenet, 1989; Malpas, 1998; Feldman and Ellenberger, 1988). Information arising from peripheral receptors and central coupling of respiratory and sympathetic neurons contribute to the rhythmic oscillations in the sympathetic activity (Bernardi et al., 2001; Häbler et al., 1996).
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Under normal conditions, sympathetic nerve activity is well correlated with phrenic discharge, exhibiting peaks of discharge mainly during the PI phase (Fig. 4A) (Malpas, 1998; Zoccal et al., 2008). This pattern of respiratory–sympathetic coupling differs from that observed during hypoxia, which is characterized by bursts of sympathetic activity mainly during the LE phase (Fig. 4B) (Dick et al., 2004; Mandel and Schreihofer, 2009). The pattern of sympathetic–respiratory coupling that occurs in response to alerting stimuli following collicular disinhibition is distinctly different from that which occurs both under normal conditions and during hypoxia. Under these conditions, the peak of sympathetic discharge occurs during the I phase (Fig. 4C). Thus, the mechanisms underlying the respiratory–sympathetic coupling following alerting stimuli must be different from to that which occurs under resting conditions and during hypoxia. It therefore seems unlikely that the observed sympatho–respiratory coupling is simply a consequence of sympathetic premotor neurons receiving a modulatory input from central respiratory neurons that in turn are activated by inputs from the colliculi. Instead, the simplest explanation for the observed respiratory–sympathetic coupling that occurs following collicular disinhibition is that both sympathetic and inspiratory premotor neurons receive inputs from a common population of command neurons. 5. Concluding remarks Our findings together with those of others suggest that command neurons within the superior and inferior colliculi play a key role in the generation of a behavioral response accompanied by synchronized cardio-respiratory changes when an animal faces a threat. Such neurons are likely inhibited by tonic GABAergic inputs from the substantia nigra pars reticulata. This subcortical network involving substantia nigra, and the midbrain colliculi appear to mediate a rapid subconscious response to threatening stimuli (Dampney, 2015). The output pathways mediating this response must be contained within the brainstem and spinal cord as the response is retained following removal of the entire forebrain. The specific interactions of these output pathways with respiratory and sympathetic premotor and motor cell groups to permit the complex pattern of responses described here remain to be determined. Grants The studies described in this article were supported by National Health and Medical Research Council of Australia (APP1028183), The Hillcrest Foundation, Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Fundac¸ão de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig) (CBB—APQ-00353-13) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (PQ30600/2013-0/472614/2011-8). References Bautista, T.G., Dutschmann, M., 2014. Inhibition of pontine Kölliker-fuse nucleus abolishes eupneic inspiratory hypoglossal motor discharge in rat. Neuroscience 267, 22–29. Bernardi, L., Porta, C., Gabutti, A., Spiazza, L., Sleight, P., 2001. Modulatory effects of respiration. Auton. Neurosci. 90, 47–56. Blanchard, D.C., Blanchard, R.J., 1972. Innate and conditioned reactions to threat in rats with amygdaloid lesions. J. Comp. Physiol. Psychol. 81, 281–290. Blanchard, R.J., Yudko, E.B., Rodgers, R.J., Blanchard, D.C., 1993. Defense system psychopharmacology: an ethological approach to the pharmacology of fear and anxiety. Behav. Brain Res. 58, 155–165. Bondarenko, E., Hodgson, D.M., Nalivaiko, E., 2014. Amygdala mediates respiratory responses to sudden arousing stimuli and to restraint stress in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 306, 951–959. Bondarenko, E., Beig, M.I., Hodgson, D.M., Braga, V.A., Nalivaiko, E., 2015. Blockade of the dorsomedial hypothalamus anf the perifornical area inhibits respiratory
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Coordinated autonomic and respiratory responses evoked by alerting stimuli: Role of the midbrain colliculi Flávia C.F. Müller-Ribeiro a,b,∗ , Ann K. Goodchild a , Simon McMullan a , Marco A.P. Fontes b , Roger A.L. Dampney c a
Australian School of Advanced Medicine, Macquarie University, NSW 2109, Australia Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais (UFMG), Minas Gerais 31270-901, Brazil c School of Medical Sciences (Physiology) and Bosch Institute, University of Sydney, NSW 2006, Australia b
a r t i c l e
i n f o
Article history: Received 7 August 2015 Received in revised form 21 October 2015 Accepted 23 October 2015 Available online 10 November 2015 Keywords: Superior colliculus Inferior colliculus Alerting response Respiratory activity Respiratory–sympathetic coupling Command neuron
a b s t r a c t Threatening stimuli trigger rapid and coordinated behavioral responses supported by cardiorespiratory changes. The midbrain colliculi can generate coordinated orienting or defensive behavioral responses, and it has been proposed that collicular neurons also generate appropriate cardiovascular and respiratory responses to support such behaviors. We have shown previously that under conditions where collicular neurons are disinhibited, coordinated cardiovascular, somatomotor and respiratory responses can be evoked independently of the cortex by auditory, visual and somatosensory stimuli. Here we report that these natural stimuli effectively increase inspiratory time most likely though phase switching. As a result the pattern of phrenic and sympathetic coupling is an inspiratory-related sympathoexcitation. We propose that blockade of tonic GABAergic input in the midbrain colliculi permits alerting stimuli to drive command neurons that generate coordinated cardiovascular, respiratory and motor outputs. The outputs of these command neurons likely interact with the central respiratory pattern generator, however the precise output pathways mediating the coordinated autonomic and respiratory responses remain to be determined. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The acute alerting response to threatening stimuli consists of an integrated and stereotyped pattern of autonomic, respiratory and somatomotor changes that increases the probability of survival and is found in all species. Interestingly, the physiological responses seen in animals exposed to diverse environmental challenges include specific stereotyped changes. A sudden alerting stimulus, usually signaled by a visual, auditory or somatosensory input, immediately generates a behavioral response such as avoidance or flight depending on the intensity of the stimulus (Dampney et al., 2008; Kabir et al., 2010). The behavioral response is always accompanied by autonomic changes characterized by increases in blood pressure (BP), tachycardia, mydriasis, piloerection and cutaneous vasoconstriction (Hilton, 1982; Dampney et al., 2008). Respiratory changes of rapid onset, which consist of increases in respiratory rate and depth, are also observed in an alerting response
∗ Corresponding author at: Instituto de Ciências Biológicas, Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, Minas Gerais, Brazil. E-mail address: fl[email protected] (F.C.F. Müller-Ribeiro). http://dx.doi.org/10.1016/j.resp.2015.10.012 1569-9048/© 2015 Elsevier B.V. All rights reserved.
(Blanchard and Blanchard, 1972; Kabir et al., 2010). The increase in arterial pressure provides perfusion of skeletal muscles priming them for activity, whereas, the increase in respiratory activity optimizes gas exchange to match increased activity or vigilance optimizing the fight/flight reaction (Dampney et al., 2008). Previous studies have indicated that sites within the central nervous system such as the dorsomedial hypothalamus, periaqueductal grey (PAG), amygdala and the midbrain colliculi are involved in the generation of coordinated aversive responses to threatening stimuli (Dampney et al., 2013; Dampney, 2015). However, the pattern of the evoked responses vary greatly with the nature of the danger; depending upon whether the threat is present and requires immediate action, or simply enhanced and sustained vigilance (Blanchard et al., 1993). 2. Role of the midbrain colliculi in the alerting response The superior colliculus (SC) is a layered region localized in the dorsal region of the midbrain. The superficial layers receive a direct innervation from the retina and an indirect one from the visual cortex, whereas the intermediate and deep layers receive auditory and somatosensory inputs (Dean et al., 1989). Electrical and chemical
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Fig. 1. Examples of recordings from two different experiments: intact brain and following decerebration. (A) Cardiovascular, respiratory and somatomotor responses evoked by pinch after microinjection of picrotoxin into the colliculus. (B) Cardiovascular, sympathetic and respiratory responses evoked by another sensory stimulus (touching whiskers) after microinjection of picrotoxin into the colliculus. (C) Cardiovascular and respiratory responses evoked by pinch after microinjection of bicuculline into the colliculus in a decerebrate preparation.
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Fig. 2. Schematic diagram showing the subcortical loop involving the superior colliculus (SC) and basal ganglia, based on a scheme proposed by McHaffie et al. (2005). The deep layers of the SC receive topographically organized visual, auditory, and somatosensory inputs, whereas, superficial layers receive only visual inputs. There are multiple parallel projections from the deep layers of the SC to the striatum, relayed via intralaminar nuclei (ILN) in the thalamus. The striatum also receives inputs from the cortex, amygdala and possibly also the dorsomedial hypothalamus (DMH), and may act as an action selector, comparing inputs from the SC with those from the cortex, amygdala and DMH. For further details see the text. The red lines indicate an excitatory projection, whereas, the blue lines indicate an inhibitory projection. Modified from Fig. 7 in Ref. Dampney (2015). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Fig. 3. (A) Example of auditory-evoked responses with SpSNA, PNA and VNA burst synchrony after microinjection of picrotoxin into the midbrain colliculi. The vertical shaded line indicates the blockade of the post inspiratory peak by the auditory stimulus. (B) Portion of the single clap response showing phrenic nerve activity overlaying vagus nerve activity in a normal breathing cycle and after auditory stimulus. SpSNA: splanchnic nerve activity, PNA: phrenic nerve activity, VNA: vagus nerve activity, I: inspiration, PI: postinspiration (stage 1 expiration), LE: late expiration (stage 2 expiration), SNA: sympathetic nerve activity.
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Fig. 4. Examples of different respiratory-sympathetic coupling patterns. (A) Respiratory-sympathetic response under resting conditions in which the peak of sympathetic activity occurs during the PI phase. (B) Phrenic-triggered averages in sympathetic nerve activity during hypoxia in which the peak of sympathetic nerve activity occurs during the LE phase. (C) Averaged sympathetic and respiratory responses to alerting stimuli following collicular disinhibition showing that increases in sympathetic activity exhibited peaks of discharge coincident with inspiratory discharge. The traces in A and B are re-drawn from Figs. 1C and 5B in Ref. Dick et al. (2004), with permission. Abbreviations: SNA: sympathetic nerve activity, PNA: phrenic nerve activity.
stimulation of the SC suggest that it is involved in the generation of two types of behavioral responses to novel sensory stimuli: an orienting response toward a stimulus or a defensive response such as avoidance or flight (Dean et al., 1989). It appears that the type of the behavioral response generated depends on the degree of threat. Lesions of the SC reduce behavioral responses to visual stimuli and indicate a major role in defensive responses (Goodale and Murison, 1975). An appropriate cardiorespiratory response would be expected to match this behavioral response as part of a coordinated response to the appearance or sound of a predator, for example. Consistent with this, increases in blood pressure, heart rate as well as respiratory rate and depth were observed following stimulation of neurons within the SC (Keay et al., 1988). The pattern of these cardiorespiratory responses varied depending on the injection site, with more pronounced responses obtained after stimulation of the intermediate and deep layers. The inferior colliculus (IC) is a well known relay station for auditory pathways in the midbrain. Similar to the SC, functional evidence supports a role in the generation of defensive responses. In particular, electrical or chemical stimulation of this region elicits behaviors characterized by alertness, freezing or escape (Brandao et al., 1993) and again the responses were accompanied by autonomic changes typically seen when the animal is confronted with a threatening stimuli (Brandao et al., 1993). Together, these studies suggest that the midbrain collicular region is capable of generating integrated behavioral and autonomic responses when appropriately triggered. In accordance with this idea, we have previously shown that disinhibition of neurons localized within the intermediate and deep layers of the superior colliculus and the central and external nuclei of the inferior colliculus in anesthetized rats, resulted in a response characterized by highly synchronized sympathetic and respiratory bursts of nerve activity, as well as increases in blood pressure and heart rate that resemble the autonomic and respiratory response triggered by alerting stimuli (Iigaya et al., 2012). If this region is involved in the generation of a coordinated cardiorespiratory response and receives auditory, visual and somatosensory inputs, it would be expected that an alerting stimulus could evoke the same pattern of response. Recently, we tested this hypothesis and showed that in anesthetized rats an auditory (sound of a clap), visual (flash of light) or somatosensory (pinch) stimulus generated immediate and
coordinated sympathetic, respiratory and somatomotor changes followed by increases in blood pressure, but only after disinhibition of neurons within the collicular region (Fig. 1A) (Müller-Ribeiro et al., 2014). In 8 experiments, a non-nociceptive somatosensory stimulus (touching whiskers) was used which also generated the same pattern of coordinated response (Fig. 1B). The responses were highly synchronized, with short intervals (20–50 ms) between the onsets of the somatomotor, respiratory and sympathetic responses with the onset of the somatomotor burst preceding the onset of the respiratory and then the sympathetic bursts. If a common population of neurons within the colliculi generates these responses, such differences in onset latencies would be expected because postganglionic sympathetic nerve fibers are unmyelinated and have a slower conduction velocity than myelinated axons of phrenic and sciatic motoneurons (Iigaya et al., 2012; Müller-Ribeiro et al., 2014). These observations led us to propose that these responses are driven by a common population of command neurons localized within the SC and IC (Müller-Ribeiro et al., 2014). Remarkably, auditory and somatosensory-evoked responses were still intact after removal of the entire forebrain (Fig. 1C). Therefore, the essential circuitry subserving these responses under conditions in which GABAergic inhibition to the collicular region is blocked in anesthetized rats, does not include the amygdala, hypothalamus, PAG, and prefrontal cortex, all regions known to mediate cardiovascular, respiratory and behavioral responses to stressful situations. At the same time, previous studies in conscious rats have shown that inhibition of the amygdala or the dorsomedial hypothalamus greatly reduces the increases in respiratory rate evoked by either a brief alerting visual or auditory stimulus or a more prolonged stressful stimulus (Bondarenko et al., 2014, 2015). A possible explanation that would reconcile our previous findings in anesthetized rats with those of Bondarenko et al. (2014, 2015) has been proposed by Dampney (2015) (Fig. 2), based on a scheme proposed by McHaffie et al. (2005). The substantia nigra pars reticulata (SNpr) is a major source of direct tonic GABAergic input to the colliculi (Redgrave et al., 1992; Castellan-Baldan et al., 2006). The SNpr itself receives inhibitory inputs from the striatum that in turn receives inputs from the deep layers of the SC relayed via the thalamus as well as inputs from the cortex and other forebrain regions (Castellan-Baldan et al., 2006; Groenewegen et al., 1999; McHaffie et al., 2005). The SNpr and striatum are components of
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the basal ganglia. The inputs to the SC from visual, auditory, and somatosensory inputs and their connections within this subcortical loop are highly topographically organized, with the result that the pattern of inputs to the striatum are highly specific for a particular stimulus (Dean et al., 1989; McHaffie et al., 2005). McHaffie et al. (2005) proposed that the striatum may act as an “action selector,” comparing inputs from the SC with those from the cortex, so that the most appropriate action can be selected for a particular set of inputs, by withdrawing inhibition from the particular output neurons in the SC that regulate the appropriate coordinated somatomotor/autonomic response. It is conceivable that direct or indirect descending pathways from the amygdala and dorsomedial hypothalamus also project to the striatum, and may be essential for the full expression of coordinated somatomotor, autonomic and respiratory changes generated by collicular neurons in response to alerting stimuli. In that case, inhibition of either the amygdala and dorsomedial hypothalamus would attenuate responses to alerting stimuli, as demonstrated by Bondarenko et al. (2014, 2015). It should also be noted that alerting stimuli in conscious rats can elicit extreme tachypnea or sniffing, with respiratory rates increasing up to 450–500 breaths/min (Bondarenko et al., 2015). Such extreme responses do not occur in anesthetized rats in response to stimulation of sites in the colliculi (Iigaya et al., 2012). This may be due to the depressant effect of anesthesia on the respiratory rate generator in the brainstem, or alternatively may reflect the fact, as stated above, that the full expression of the respiratory response to alerting stimuli depends upon structures in the forebrain as well as the colliculi. What output pathways from the colliculi could mediate the autonomic, respiratory and behavioral responses? The output pathways from the SC have been studied primarily with regard to motor function. Projections from the midbrain colliculi include a projection to the dorsolateral periaqueductal grey (PAG) (Rhoades et al., 1989), which regulates sympathetic and respiratory activity probably via ascending projections to the dorsomedial hypothalamus (de Menezes et al., 2009; Horiuchi et al., 2009). Although PAG and SC share some anatomical and functional properties, the circuitry mediating these responses seems to be different. There are also descending projections from the SC to the pontomedullary reticular nuclei, but these do not appear to target the rostral ventrolateral medulla or the dorsal and ventral respiratory groups, regions known to control sympathetic and respiratory activity (Redgrave et al., 1987). Similar, the neural pathways of the IC involved in generating the sympathetic, respiratory and somatomotor responses are still poorly understood. There is also a direct projection from the IC to the SC (Garcia del Cano et al., 2006) and it is conceivable that this connection may target neurons in the SC that evoke sympathetic, respiratory and somatomotor response. It is unclear, however, how the autonomic and respiratory output is processed and how this output is coordinated with the motor output.
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the PI or LE phase, the expiratory period was shortened, however, if the stimulus arrived during inspiration the duration of the inspiratory activity was either unchanged or increased but with little effect on the duration of the expiratory period (e.g., Fig. 7A in Ref. Müller-Ribeiro et al., 2014). In one experiment in that study, we also measured the vagal nerve activity (VNA) in addition to PNA, to observe more directly the effects on I, PI and LE. Fig. 3 shows examples of the respiratory responses evoked by auditory stimuli (after blockade of GABA receptors in the colliculi) in this experiment. Each stimulus selectively blocked the post inspiratory peak in the vagus nerve (VNA) effectively shortening the subsequent expiratory period as can be seen in the recordings of PNA and VNA (Fig. 3). In this experiment, there were 103 respiratory cycles in which the pattern of VNA activity was altered following a stimulus, and in which the 3 phases of the respiratory cycle could be identified from the VNA recording. Out of these 103 cycles, the PI peak was abolished in 58 cases (56%) and decreased in the remaining 45 cycles (44% of the total). In contrast, the stimulus had little effect on the I peak (unchanged in 87 cases, increased in 10 cases and decreased in 6 cases). Statistical analysis revealed that these different effects on the PI and I peak were highly statistically significant (Chi-squared test, P < 0.001). Such a selective effect on the PI component of the respiratory cycle has only been generated from three regions, all located in the brainstem: the Kölliker-fuse nucleus, Bötzinger complex and the nucleus tractus solitarius (NTS) (Dutschmann et al., 2014). Inhibition or removal of the Kölliker-fuse nucleus evokes a similar pattern of activity in the vagus nerve (Smith et al., 2007; Bautista and Dutschmann, 2014; Dutschmann and Herbert, 2006). Injection of the inhibitory peptide somatostatin in the Bötzinger complex selectively abolishes post inspiratory activity (Burke et al., 2010) whereas, blockade of glutamate receptors in the (NTS) reduces postinspiratory activity (Costa-Silva et al., 2010). It is therefore possible that one or more of these candidate regions may receive inputs from the midbrain colliculi which may modify timing or phase switching of the respiratory cycle. However, the respiratory effect following sensory stimuli after disinhibition of the colliculus was dependent upon the phase of the respiratory cycle in which the stimulus arrived. The inhibition of postinspiratory activity occurred when the stimulus arrived during LE. If the stimulus arrived during PI the expiratory period immediately following was also shortened (Fig. 1C) however, when the stimulus arrived during inspiration the inspiratory activity was lengthened and there was little effect on the expiratory period (Fig. 1A). Additional examples of this phase switching are also evident in our previously published work (Müller-Ribeiro et al., 2014, Fig. 7). All of these patterns effectively increase the period of inspiration when the animal receives an alerting stimulus. These data strongly indicate that the output pathways activated by the stimuli must interact with the central pattern generator. The inhibition of the PI component decreases the expiratory period and thus increases respiratory frequency when the animal receives an alerting stimulus.
3. Effects on the respiratory pattern generator of natural stimuli mediated by the colliculi The central pattern generator comprising premotor respiratory neurons in pontomedullary locations (Bautista and Dutschmann, 2014) produces a breathing cycle composed of 3 phases: inspiration (I), post inspiration (PI) and late expiration (LE) (Fig. 3A), which can be identified in the integrated activity of phrenic and vagus nerves (Richter, 1996; Richter and Spyer, 2001). In our previously published study (Müller-Ribeiro et al., 2014), we observed that when stimuli evoked respiratory responses following collicular disinhibition, the effect on the pattern of phrenic activity (PNA) depended on the timing of the stimulus with respect to the phase of the respiratory cycle. In particular, if the stimulus arrived during
4. Pattern of respiratory–sympathetic coupling generated by inputs from the colliculi Sympathetic nerve activity is modulated by the respiratory system via connections from respiratory neurons in the lower brainstem to sympathetic pre motor neurons (Haselton and Guyenet, 1989; Malpas, 1998; Feldman and Ellenberger, 1988). Information arising from peripheral receptors and central coupling of respiratory and sympathetic neurons contribute to the rhythmic oscillations in the sympathetic activity (Bernardi et al., 2001; Häbler et al., 1996).
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Under normal conditions, sympathetic nerve activity is well correlated with phrenic discharge, exhibiting peaks of discharge mainly during the PI phase (Fig. 4A) (Malpas, 1998; Zoccal et al., 2008). This pattern of respiratory–sympathetic coupling differs from that observed during hypoxia, which is characterized by bursts of sympathetic activity mainly during the LE phase (Fig. 4B) (Dick et al., 2004; Mandel and Schreihofer, 2009). The pattern of sympathetic–respiratory coupling that occurs in response to alerting stimuli following collicular disinhibition is distinctly different from that which occurs both under normal conditions and during hypoxia. Under these conditions, the peak of sympathetic discharge occurs during the I phase (Fig. 4C). Thus, the mechanisms underlying the respiratory–sympathetic coupling following alerting stimuli must be different from to that which occurs under resting conditions and during hypoxia. It therefore seems unlikely that the observed sympatho–respiratory coupling is simply a consequence of sympathetic premotor neurons receiving a modulatory input from central respiratory neurons that in turn are activated by inputs from the colliculi. Instead, the simplest explanation for the observed respiratory–sympathetic coupling that occurs following collicular disinhibition is that both sympathetic and inspiratory premotor neurons receive inputs from a common population of command neurons. 5. Concluding remarks Our findings together with those of others suggest that command neurons within the superior and inferior colliculi play a key role in the generation of a behavioral response accompanied by synchronized cardio-respiratory changes when an animal faces a threat. Such neurons are likely inhibited by tonic GABAergic inputs from the substantia nigra pars reticulata. This subcortical network involving substantia nigra, and the midbrain colliculi appear to mediate a rapid subconscious response to threatening stimuli (Dampney, 2015). The output pathways mediating this response must be contained within the brainstem and spinal cord as the response is retained following removal of the entire forebrain. The specific interactions of these output pathways with respiratory and sympathetic premotor and motor cell groups to permit the complex pattern of responses described here remain to be determined. Grants The studies described in this article were supported by National Health and Medical Research Council of Australia (APP1028183), The Hillcrest Foundation, Coordenac¸ão de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Fundac¸ão de Amparo à Pesquisa do Estado de Minas Gerais (Fapemig) (CBB—APQ-00353-13) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (PQ30600/2013-0/472614/2011-8). References Bautista, T.G., Dutschmann, M., 2014. Inhibition of pontine Kölliker-fuse nucleus abolishes eupneic inspiratory hypoglossal motor discharge in rat. Neuroscience 267, 22–29. Bernardi, L., Porta, C., Gabutti, A., Spiazza, L., Sleight, P., 2001. Modulatory effects of respiration. Auton. Neurosci. 90, 47–56. Blanchard, D.C., Blanchard, R.J., 1972. Innate and conditioned reactions to threat in rats with amygdaloid lesions. J. Comp. Physiol. Psychol. 81, 281–290. Blanchard, R.J., Yudko, E.B., Rodgers, R.J., Blanchard, D.C., 1993. Defense system psychopharmacology: an ethological approach to the pharmacology of fear and anxiety. Behav. Brain Res. 58, 155–165. Bondarenko, E., Hodgson, D.M., Nalivaiko, E., 2014. Amygdala mediates respiratory responses to sudden arousing stimuli and to restraint stress in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 306, 951–959. Bondarenko, E., Beig, M.I., Hodgson, D.M., Braga, V.A., Nalivaiko, E., 2015. Blockade of the dorsomedial hypothalamus anf the perifornical area inhibits respiratory
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