All methods and procedures were approved by the Drexel University Institutional Animal Care and Use Committee, which oversees Drexel University’s AAALAC International-accredited animal program. Experiments were conducted upon spontaneously-breathing Sprague-Dawley adult male rats (330-480 g) anesthetized with isoflurane (Matrix; 4-5% induction, 2.0-2.5% maintenance) vaporized in hyperoxia (97.5-98% fractional inspiration of O₂) via a snout mask. We recorded electrocardiogram (EKG) activity via three small subcutaneous electrodes placed in the classic Einthoven positions using conventional amplification and filtering settings (Neurolog; Digitimer, Hertfordshire, United Kingdom) and monitored using audio amplification (model AM10; Grass Instruments) and an oscilloscope. We maintained anesthetic depth at a level such that limb withdrawal reflexes and changes in sinoatrial nodal period in response to pinches of the distal hindlimb were eliminated. Following incision of the tracheal cartilage using electrocautery and tracheo-luminal cannulation using an atraumatic glass tube, animals were mechanically ventilated (respiratory rate: 45-65 cycles/min; tidal volume: 2.5-3.0 mL per breath; Columbus Apparatus) using the same gas mixture (Table 1). Cannulae were introduced into the right femoral artery and vein in order to record arterial pressure and intravenously infuse fluids and drugs, respectively. During the initial general surgical preparation and neural recordings, we maintained rectal temperature at 37.0 ± 0.1 °C through the use of the surgical lamp. The phrenic nerves were accessed and dissected from the brachial plexus and investing translucent fascia via a cervical pre-scalene approach. We visually identified the common carotid, external carotid, and internal carotid arteries and dissected investing soft tissue encasing the bifurcation of the common carotid artery in the high cervical region (Fig. 10). Soft tissue investing the vessels was gently incised, separated, and removed using sharp and fine microdissection. We identified the proximal extent of the occipital artery emanating from the proximal extent of the external carotid artery and clearly distinguished it from the proximal extent of the internal carotid artery. We ligated the internal carotid arteries bilaterally rostrally with respect to the origins of the pterygopalatine artery effectively preserving the integrity of the baroreceptor-rich carotid sinuses and chemoreceptor-rich carotid bodies (Fig. 11). We occasionally reapproximated the precervical soft tissues in anatomic layers utilizing continuous 6-0 silk suture. We reapproximated the ventral midline rostrocaudally-oriented midline cervical incision utilizing continuous 4-0 silk suture. We subsequently situated the animal prone in a stereotaxic device and placed lateromedially mobilizable titanium bars in the external acoustic meati bilaterally to achieve head fixation.

Rigid head fixation allows the surgeon to trephine the animal under mechanically stable conditions, though should not be excessively overzealous to an extent which significantly compromises presympathetic bulbospinal excitatory synaptic drive conveyed to somatodendritic neuronal membranes of intermediolateral cell column preganglionic sympathetic neurons monosynaptically and polysynaptically through interposed interneuronal relays and generate consequent reductions of the arterial blood pressure. Bilateral everting scalp retraction utilizing 4-0 silk sutures placed through the cutis and subcutis follows rostrocaudally oriented sagitally-approximated incision using scissors (Fig. 12). Microdissection of epicranial soft tissue (Fig. 13) proceeds utilizing blunt instruments and that surrounding the margins of the cranial convexity proceeds utilizing bipolar electrocautery (Fig. 14). A diamond drill bit generates generous biparietal craniectomy windows leaving a narrow segment of medianly situated overlying the parietal extent of the superior sagittal sinus (Figs. 15-19). We microsurgically carve concave cuts in the narrow segment of bone overlying the superior sagittal sinus lateromedially using a smaller diamond bit at the four angles between the superior sagittal sinus (SSS)-adjacent the medial sagittal and caudal transverse margins bordering the craniectomy windows and in order to thin the area, thus rendering continuous the two sets of bays (rostral and caudal) (Fig. 18). We subsequently sharply incise the variably tense dura mater covering the left and right parietal lobes of the cerebral hemispheres. Some bleeding may ensue from transection of the dural vessels, though should not alarm the veteran and seasoned investigator (Fig. 19) and may be effectively curtailed by gently placing a cold thrombin-soaked gelfoam sponge on the oozing region and sufficiently suspend the microsurgical impulse to permit the native therapeutically-activated procoagulant pathways to generate platelet-fibrin thrombi in the transected ends of the fine dural vessels. We subsequently thread a 6-0 or 4-0 silk suture needle through one parietal lobe, beneath the superior sagittal and paired anterior cerebral arteries, through the falx cerebri, and ventrodorsally through the substance and surface of the contralateral parietal lobe (Fig. 19). We use the same thread to ligate the superior sagittal sinus to the overlying narrow median segment of bone in the bay areas of thinned cancellous and compact calvarial bone. We repeat this maneuver in order to rostrally and caudally ligate the superior sagittal sinus.

At this juncture, the remainder of the procedure may take on one of several permutative variations. Osteotomy of the narrow median segment of bone overlying the superior sagittal sinus between the ligatures may be performed and the interposed osseous wedge microsurgically drilled using a diamond drill bit connected to a high-speed surgical drill and removed. Rostral and caudal ligation of the superior sagittal sinus (Fig. 20) permits transective interruption utilizing iridectomy scissors (Fig. 21). Alternatively, the bone overlying the superior sagittal sinus may be left in place. The former strategy facilitates decerebrative encephalotomy and the latter marginally reduces operative time. When performing osteotomy and double ligation and transection of the SSS, diencephalo-mesencephalic transection may be performed via a single motion by insertively advancing a microspatula into cerebral parenchyma at the supracollicular level approximately 2.0 to 2.5 mm rostral to the level of lambda and carrying the transection laterally across the midline (akin to cutting cake), while avoiding severing the posterior communicating arteries at the cranial base (Fig. 22). When maintaining the narrow median segment of bone overlying the superior sagittal sinus in place, two motions are consequently required in order to successfully perform the encephalotomy - the first motion advances the microspatula through one craniectomy window underneath the superior sagittal sinus into the contralateral posteroinferolateral sphenosquamous suture between the middle and posterior cranial fossae, then carrying the transection ipsilaterally using the same motion repeated through the contralateral parietal craniectomy window.

Removal of supratentorial cerebral tissue rostral to the transverse rostral transected surface of the mesencephalon via aspirative suctioning follows encephalotomy. Temporal, frontal, and occipital lobectomies follows incipient aspirative parietal lobectomy (Fig. 22). Aspiration of the occipital lobe proceeds sans injury to the rhombencephalon by maintaining the suction tip at the most superficial aspect of the caudal margins of the parietal craniectomy windows. The anterior and middle cranial fossae are packed with gelfoam soaked in cold thrombin solution following suctioning of the cerebral parenchyma (Fig. 23). Underpacking of the cranial cavity should be avoided in order to ensure effective skull base hemostasis and mechanically preclude rebleeding and overpacking of the cranial cavity should be avoided in order to prevent transforaminal herniation of the parenchymal contents of the posterior cranial fossa (Fig. 24). Initial post-decerebrative recovery of animal arterial blood pressure, sympathetic activity, and neural network synchrony proceeds at the same anesthetic depth. Weaning and eventual withdrawal of anesthesia should proceed slowly during the course of approximately one hour to the extent permitted by hemodynamic condition. We typically reduce the level of isoflurane anesthesia in quantal decrements of approximately 0.5% every 15 to 20 minutes, though we encourage the custom design and individualization of perioperative management of anesthetics fitting the instinct of the investigator. The incipient development of "neurophysiologist instinct" typically requires one to have extensively conducted neural recordings in decerebrate animal preparations. Prior to complete withdrawal of isoflurane anesthesia, we characteristically administer a bolus injection of 2 mg/kg vecuronium bromide (0.4 mg/mL) followed by intravenous infusion (4 mg kg^-1 h^-1) of the same alternately dissolved in Ringer-Locke solution or artificial cerebrospinal fluid. Preservation of parenchymal between the mesencephalic locomotor region, (comprised of pedunculopontine and cuneiform nuclei), and medullary reticulospinal units may permit locomotor activity in the unanesthetized decerebrate condition. We have occasionally observed forelimb and hindlimb locomotor activity representing nascent soi-disant "attempts" at running in the event of anesthetic weaning sans commensurate administration of neuromuscular-type nicotinic acetylcholinergic antagonist [Nota Bene: We have used vecuronium to achieve effective neuromuscular antagonism and flaccid paresis, though the effects of rocuronium appear to wear off extremely rapidly in our experimental preparation. We do not have a satisfactory nor adequate way to explain this, though we posit rapid metabolism or ligand-receptor mismatching could putatively prevent rocuronium from effectively achieving neuromuscular antagonism sufficient to permit neuronal and neural recordings in the unanesthetized preparation of the decerebrate rat].

Decerebration was successfully performed in all animals sans bleeding or neurogenic shock refractory to resuscitative maneuvers. Arterial blood pressure recovered to normotensive or slightly hypertensive values in all animals following decerebration (Fig. 2). Electrophysiological recordings and experimental interventions were typically conducted 1 to 2 hours following decerebrative encephalotomy. Neural recordings of hypoglossal and phrenic nerve efferent activity exhibiting a high ratio between the spatiotemporally dynamic amplitude of rhythmic phrenic bursting and background tonic noise were successfully obtained across all experiments (Fig. 3). Monophasic recordings of hypoglossal and phrenic neural efferent activity revealed regularly rhythmic bursting synchronized with the ventilator cycle. Hypoglossal neural efferent activity demonstrated regularly rhythmic bursting and an augmenting pattern of preinspiratory inspiratory discharge and phrenic neural efferent activity evidenced an augmenting pattern of inspiratory discharge, with variable postinspiratory activity. We venture to propose the presence of an augmenting pattern of phrenic nerve discharge represents an unparalleled metric indicating integrity of brainstem propriobulbar and bulbospinal circuitry.

In our experimental experience, we have observed several general trends correlating with successful procedural outcome which may prove beneficial and instructive to the investigator seeking to optimize and adapt the technique of decerebration in neurophysiological experimentation (Table 2). We recommend induction and maintenance of animals using potent inhalational anesthetics during the general surgical preparation, since the distribution of fat-soluble agents including pentobarbital, urethane, or alpha-chloralose to adipose stores and biphasic elimination, comprised of replace with Greek letter alpha (a) (fast) and replace with Greek letter beta (b) (slow) phases, could render decerebrate models prepared using these agents, in effect, slightly anesthetized. We have critically observed that maintaining relative hypothermia, with a core body temperature of approximately 32.5-35.5 °C, during biparietal trephination, decerebrative encephalotomy, skull base hemostasis, and postprocedural recovery generates more stable animal preparations compared with normothermic ( > 36 °C) conditions. It may be that moderate hypothermia protects the neuronal and astrocytic elements constituting the brainstem during the neurogenic hypotension which immediately follows decerebrative transection (Shintani et al., 2010). Concurrent use of a heating blanket and the surgical lamp often proves sufficient in maintaining normothermia during the operation. Accordingly, during the initial surgical preparation, exclusive use of the surgical lamp sans heating blanket typically generates moderate neuroprotective hypothermia. Following decerebration, we utilize a servo-controlled heating blanket in order to maintain animal temperature within 37.0 ± 0.5 °C.

Rigidity of head fixation occasionally correlates inversely with magnitude of the arterial pressure: tightening the head fixation reduces blood pressure and gently releasing it slightly systolic, and diastolic, arterial blood pressure. Accordingly, the animal’s head should be fixed to an extent permitting craniotomies to be performed in a mechanically stable manner, though not excessively so such that animal systolic arterial pressure decreases below 90 mmHg nor diastolic arterial pressure decreases below 60 mmHg.

Maintenance of lower levels of systolic arterial blood pressure (70 to 100 mmHg) during the interim period interposed between biparietal trephination through perioperative recovery may effectively reduce post-transection bleeding from the circle of Willis. However, we have typically observed greater degrees of systolic hypotension during the decerebration procedure consistently correlate with lower levels of blood pressure experienced by the animal during post-transection neurogenic shock. We accordingly believe the severity and duration of this transient hypotension could inversely correlate with animal longevity post-operatively, though certainly mitigated by the judicious use of neuroprotective hypothermia and isoflurane gas anesthesia.

Bilateral ligation of the internal carotid arteries achieves excellent hemostasis and prevents intraoperative and postoperative blood loss (Ghali and Marchenko, 2015, 2016a,b; Ghali, 2015, 2019a,c; Hayashi, 2003; Marchenko et al., 2012; Sapru and Krieger, 1978). Our use of bilateral internal carotid artery ligation accordingly carries the risk of possible ischemogenic cerebral edema, which may be mitigated by reducing, to the extent permissibly feasible, the interval interposed between vascular ligation and decerebrative transective encephalotomy. Dobson and Harris (2012) advocate the use of reversible temporary clipping of the common carotid arteries in order to protect the influence of carotid sinus baroreceptor stretch upon the activity of the sympathetic oscillators and cardiovagal premotoneurons residing within the brainstem and regulating the arterial blood pressure and sinoatrial period (Tang et al., 2010). In our experience, dissection and high ligation of the internal carotid arteries above the level of the pterygopalatine artery achieves these goals commensurately. In their technique performing decerebration in rabbits, Taylor and colleagues (1991) describe leaving one carotid artery intact in order to reduce the frequency of paroxysmal hypotensive episodes, which could putatively ensue from loss of hypothalamic neuronal inputs to rostral ventrolateral medullary presympathetic units. We accordingly suggest silk suture ligation of the internal carotid arteries above the level of the pterygopalatine artery bilaterally (Ghali and Marchenko, 2015, 2016a,b; Ghali, 2015, 2019a,c; Marchenko et al., 2012, 2015) or reversible temporary clipping of the internal carotid arteries to be performed in conjunction with packing of the skull base and cranial cavity with cold thrombin-soaked gelfoam packing without or with concurrent use of tissue adhesive represent equivalently viable alternatives to achieving reduction of cranial arterial inflow (Dobson and Harris, 2012). Though studies have occasionally anecdotally described arterial blood pressure dynamics prior to, and following, decerebration (Faber et al., 1982; Sapru and Krieger, 1978), no study has sought to characterize time variant recovery of hemodynamic variables during the interregnum. Sparing of the mesencephalic locomotor region constituted by the pedunculopontine and cuneiform nuclei by a supracollicularly placed transective encephalotomy permits animal locomotor activity in response to noxious or non-noxious cutaneovisceral stimuli. Parenteral intravenous administration of bolus and maintenance doses of neuromuscular type nicotinic acetylcholinergic antagonists prevents animal locomotor rhythmic activity, effectively permitting the neurophysiologist to successfully obtain stable supraspinal or spinal individual and/or multiunit neuronal and/or neural efferent recordings; (Marchenko et al., 2002). Light sedation renders muscle excursions sufficiently diminutive permitting the elegant conduct of electromyogram recordings. Superior sagittal sinus ligation successfully and preemptively achieves cerebral venous hemostasis effectively facilitating the execution of decerebrative encephalotomy in a single methodical transective motion using the microspatula and prevents cranial bleeding following diencephalomesencephalic transection.

There exist a variety of approaches by which the cerebrum may be exposed in order to achieve access permissive of a decerebrative transection. Prior silk suture ligation or temporary clip occlusion of the internal carotid arteries effectively reduce decerebrative transection-related bleeding and are commonly requisite among all described methods (Figs. 25 and 26). We ligate the right and left internal carotid arteries above the level of the pterygopalatine arteries within minutes of each other and within the hour preceding scalp incision preparing the cranial surface for trephination and the cerebral hemispheric convexities for decerebration. Should the time lapse interposed between internal carotid artery ligation and diencephalomesencephalic decerebrative transection prove excessive, initial animal blood pressure decreases more profoundly and animals are less hemodynamically stable following the procedure. A blood flow steal phenomenon may arise in the circle of Willis, whereby the elimination of internal carotid artery blood flow preferentially diverts basilar artery blood flow from the parenchymal contents contained within the confines of the infratentorial fossa to the parenchymal contents contained within the confines of the supratentorial fossa, accordingly and predictably generating powerful reductions in brainstem perfusion. Studies have accordingly described a myriad of variations of trephination techniques utilized to achieve operative exposure of the cranial cavity to perform decerebrative transection. One may fashion a single craniotomy inclusive of, and extending across, the left and right parietal bony convexities, gently lifting the bone from the underlying superior sagittal sinus. Avulsion of the superior sagittal sinus when significantly adherent to endosteum significantly enhances the risk of iatrogenically precipitating hemodynamically-catastrophic bleeding, especially in older animals. Accordingly, we prefer to trephine the parietal convexities bilaterally, initially preserving a 1 mm-wide strip of bone overlying the superior sagittal sinus (SSS) from bregma to lambda (Fig. 1E-1H). We generally leave a segment of bone overlying the superior sagittal sinus significantly of significantly narrower width compared with investigators and drill osteotomy bays into the sagittal strip of bone overlying the superior sagittal sinus where the superior sagittal sinus will be silk suture ligated in order to achieve better purchase, securing, and compression of this venous structure against the bone. This operative step fundamentally distinguishes our approach from the experience of the majority of investigators. Thread ligation across a broad segment of midsagittal bone would only gently compress the ventral surface of the superior sagittal sinus upwards and consequently fails to adequately secure the venous channel given the relationship between the curvature of the midsagittal bone with respect to that of the superior sagittal sinus. Ensuring the width of the midline strip of bone overlying the sagittal suture remains narrow to the greatest extent reasonably feasible yields the greatest likelihood of effectively achieving a secure ligation.

The specific technique of decerebrative encephalotomy varies according to the type of trephination employed. When biparietal craniectomies with an intact intervening median segment of sagittal bone represent our cranial access, we advance the microspatula through one craniectomy window in a manner directed contralaterally to the deepest and furthest reaches of the posteroinferolateral middle cranial fossa perpendicular to a coronal plane bisecting the mesencephalodiencephalic junction 2.0 to 2.5 mm rostral to a vertical plane through lambda and perpendicular to a transverse plane through the external acoustic meati and advanced ipsilaterally to the ipsilateral posteroinferolateral reaches of the middle cranial fossa towards the side of the craniectomy window across the skull base and repeat the same motion through the contralateral craniectomy window in order to achieve supracollicular decerebrative encephalotomy. Should biparietal craniectomies fashioned on alternate sides of the superior sagittal sinus with an intact intervening segment of bone be subsequently removed or a single trans-sagittal craniotomy inclusive of right and left halves of the parieto-cranial calvarium and extending across the perisagittal suture segment of bone overlying the superior sagittal sinus represent our cranial access, only a single transective cut gently carried from the contralateral to the ipsilateral posteroinferolateral reaches of the middle cranial fossa and across the skull base proves necessary in order to effectively dissociate the telencephalic and diencephalic elements of the cerebral hemispheres from the bulb.

Following decerebrative transective encephalotomy, we rapidly pack the anterior and middle cranial fossae using cold thrombin-soaked gelfoam sponges. We avoid both underpacking, which may achieve inadequate hemostasis and yield a persistent low rate of bleeding, and overpacking, which may precipitate transforaminal herniation. Post-transective neurogenic hypotension typically resolves within approximately one hour, in parallel with animal bolusing with, and maintenance on, intravenously administered vecuronium, with isoflurane anesthesia concurrently, gradually, and judiciously weaned. Studies have accordingly described or advocated the use of packing the cranial vault using gelatin sponges or cotton balls soaked in cold thrombin solution (de Almeida et al., 2010) and/or tissue adhesive (Dobson and Harris, 2012) in order to prevent blood loss and mechanically secure vessels transected at the base of the cranial cavity (Dobson and Harris, 2012). Some studies have accordingly relied significantly or exclusively on the use of cold thrombin-soaked gelfoam packing of the cranial fossae to achieve hemostasis. We use this technique as an auxiliary decorative measure, or icing on the cake in a manner of reference, and accordingly recommend this should not represent the principal method of achieving hemostasis. Secure and firm ligation of the internal carotid arteries bilaterally and superior sagittal sinus rostrally and caudally accordingly represent the most effective and sure methods of preventing peri-decerebrative bleeding. Thus, though some studies have described the utility of minimizing parenchymaml removal in reducing blood loss (Faber et al., 1982), we reiterate and emphasize properly securing the inflow and outflow vessels of the intracranium far outweighs any reasonably conceivable benefit derived from the collectively proposed adjunctive methods.

We will accordingly provide a few examples in order to highlight and underscore the preferability of employing unanesthetized decerebrate preparations compared with the use of anesthetized animals (Ghali and Marchenko, 2015, 2016a,b; Ghali, 2015, 2019a,c; Marchenko et al., 2012; Marchenko and Rogers, 2006a,b, 2007, 2009). Prior to our work, the highest firing rate of phrenic motoneurons recorded was shown to correspond with discharge frequencies exhibited by the medium frequency spectral band of oscillations (Christakos et al., 1991), thus leading various investigators to suggest the high frequency band of synchronized oscillatory activity extant within respiratory related neural spectra may be generated by an epiphenomenological mechanism whereby cycle skipping and summation of the medium frequency band of activity summates to higher frequencies of activity (van Brederode and Berger, 2008). Studies accordingly generated smoothed pseudo Wigner-Ville distribution (SPWVD) time frequency representations of coherence (de Souza Neto et al., 2001; Djebbari and Bereksi Reguig, 2013; Rajshekhar et al., 2009) between semicosine wave transformed unitary discharge of phrenic motoneurons and whole phrenic nerve discharge (Marchenko et al., 2012). We segregated phrenic motoneurons segregating into high, medium, and low frequency groups (Marchenko et al., 2012) correlated with the high, medium and low frequency oscillations in phrenic nerve discharge (Lebedev and Nelson, 1996) using K-means cluster analysis. Accordingly, using the unanesthetized decerebrate adult rat, we provided the first citable evidence of phrenic motoneurons discharging at rates corresponding with peak frequencies attained by high frequency oscillations in the phrenic neurogram (Fig. 1) (Marchenko et al., 2012). Our results powerfully support the hypothesis and derivative conjectures that the synchronous discharge of motoneurons at analogous frequency bands generates high frequency oscillations in phrenic nerve and respiratory related neurogram discharge (Marchenko et al., 2012). Definitively and incontrovertibly establishing this hypothesis would accordingly require contemporaneous recordings of multiple phrenic motoneurons within the phrenic motoneuron pool and demonstrating unit-to-unit and unit-to-nerve coherence at the corresponding phrenic spectral bands (personal communication V. Marchenko and R. Rogers).

Use of the unanesthetized decerebrate preparation of the adult rat has also allowed us to precisely interrogate vagal and hypercarbic modulation of brainstem neural networks generating preinspiratory activity evident in the hypoglossal nerve discharge (Fig. 2) (Ghali and Marchenko, 2016b; Ghali, 2015). Specifically, we demonstrated the presence of hypoglossal preinspiratory discharge in the vagus intact condition in the unanesthetized decerebrate preparation of the adult rat (Ghali, 2015), controverting the findings of Fukuda in the anesthetized preparation of the adult rat, and vagal accentuation of the activity (Ghali and Marchenko, 2016b; Ghali, 2015). We demonstrated potent crossmodal modulation of hypoglossal preinspiratory and inspiratory responses to hypercapnia by intactness of the vagal nerve. Accordingly, we observed greater accentuations of both the preinspiratory and inspiratory components of the respiratory neuromotor output in response to hypercarbia in the vagotomized compared with the vagus intact state (Ghali and Marchenko, 2016b).

Supraphrenic hemisection classically completely abolishes activity in the phrenic nerve discharge ipsilateral to the lesion with variable rapidity of recovery (Ghali, 2017d). Porter (1895) initially demonstrated cessation of ipsilateral diaphragmatic chest wall movements following high cervical hemisection above the level of the phrenic nucleus in morphine anesthetized preparations of a dog and several anesthetized rabbits as. Transective division of the phrenic nerve contralateral to the side of spinal cord hemisection effectively resurrected diaphragmatic and intercostal activity ipsilateral to spinal cord lesioning, evidenced by the reemergence of chest wall movements (Porter, 1895). Studies have accordingly termed and designated this pattern of recovery the soi-disant crossed phrenic phenomenon, whereby a stressor provoking the accentuation of neural discharge by propriobulbar neuronal circuitry constituting the respiatory central pattern generator elicits reemergence of respiratory related neural activity evident in neurogram, electromyogram, or chest wall movements, ipsilateral to a supraphrenic hemisection of the spinal cord (Ghali and Marchenko, 2015; Ghali, 2017d). Investigators would later demonstrate the same phenomenon of reemergence of the phrenic motoneuronal population activity, as demonstrated by diaphragmatic electromyography (Goshgarian, 1979, 1981; Gransee et al., 2017; Martinez-Galvez et al., 2016) and phrenic electroneurography (Fuller et al., 2006, 2008; Golder et al., 2003; Golder and Mitchell, 2005; O'Hara and Goshgarian, 1991; Polentes et al., 2004; Zimmer and Goshgarian, 2007) in response to hypercapnia, hypoxia, or contralateral phrenicotomy. Studies have also since successfully demonstrated spontaneous recovery of activity in the phrenic nerve discharge ipsilateral to the site of lesioning, with earlier recovery observed when utilizing phrenic electroneurographic (Fuller et al., 2006; Golder and Mitchell, 2005; Zimmer and Goshgarian, 2005, 2006, 2007), compared with diaphragmatic electromyographic (Goshgarian, 1981; Martinez-Galvez et al., 2016), evaluation of the phrenic motoneuronal population activity. Studies mechanistically attributed the observed patterns of recovery following cervical spinal cord hemisective injury to reemergence of latent descending crossed phrenic pathways decussating below the level of the lesion or the ingrowth of axons to the contralateral phrenic motor nucleus (Ghali and Marchenko, 2015; Ghali, 2017a). Since the use of anesthesia attenuates neural respiratory outputs, we hypothesized more rapid recovery of spontaneous crossed phrenic nerve activity would be revealed through the use of unanesthetized models. We successfully identified spontaneous recovery of crossed activity in the phrenic nerve discharge ipsilateral to a C₁ hemisection in the unanesthetized preparation of the supracollicularly decerebrate adult rat commencing within minutes and progressing during the course of hours (Fig. 3), summarily controverting and overturning the findings of Porter (1895) and his successors (see Tables 1 and 2 of Ghali (2017a)). Readministration of isoflurane anesthesia in the decerebrate condition effectively annihilated recovery in phrenic nerve discharge ipsilateral to C₁ hemisection (Fig. 4), supporting our hypothesis crossed polysynaptic bulbophrenic pathways may be constitutively active and preferentially suppressed by anesthesia and spinal shock.

Studies have provided robust evidence across a variety of species and animal models indicating the spinal cord may be capable of generating rhythmic activity distributing to respiratory related neural outputs (Aoki et al., 1980; Palisses et al., 1988, 1989; Reinoso et al., 1996; Viala et al., 1979). Spontaneous recovery of respiratory related neural activity was previously demonstrated to occur in cats (Aoki et al., 1980) and dogs (Reinoso et al., 1996). The works of Viala and colleagues identified pharmacologically induced respiratory-locomotor rhythmic activity across several studies performed in various preparations of the rabbit (Corio et al., 1993; Dubayle and Viala, 1996; Palisses et al., 1988, 1989). Utilizing the unanesthetized decerebrate preparation of the Sprague-Dawley adult rat, we demonstrated spontaneous, pharmacologically-generated, and asphyxia induced patterns of bilaterally synchronized rhythmic bursting activity in phrenic nerve discharge following bulbospinal interruption achieved via a transective injury through a plane between the brainstem and cervical cord at the C₁ spinal level (Ghali and Marchenko, 2016a). Patterns of spontaneous activity following C₁ transection included pseudoregular rhythmic bursting (Fig. 5) and slow synchronous oscillations (Ghali and Marchenko, 2016a), with oscillatory frequency variably consistent with that of the vasogenic autorhythmicity. Chemical disinhibition of upper cervical (C₁ to C₂) phrenic interneurons using topical application of a cocktail containing a mixture of gabazine (4 mM) and strychnine (4 mM) generated bilaterally synchronized decrementing bursts of variable duration exhibiting maximal peak of discharge during onset, bearing striking resemblance to prolonged gasps evident in bulbospinal intact preparations (Fig. 6) (Ghali and Marchenko, 2016a). Asphyxic challenge generated bilaterally synchronized decrementing rhythmic bursting evident in phrenic nerve discharge in C₁ transected unanesthetized decerebrate rats (Fig. 7) (Ghali and Marchenko, 2016a), suggesting the existence of propriospinal circuitry constituting a spinal gasping generator localizing to the upper cervical spinal cord (Fig. 8).

In the 19^th century, Dittmar (1873) demonstrated precipitous declines of the arterial pressure following cervical transection dissociating the bulb from spinal cord preganglionic sympathetic neurons. Investigators have extensively and consistently demonstrated profound reductions of the arterial pressure following high cervical transection followed by variable recovery to near normal values occurring between several days to weeks following injury, a demonstration seminally made by Sherrington (1906) and replicated by the experience of subsequent studies (Burke, 2007; Dittmar, 1873; Goltz, 1874; Sherrington, 1906). Using the unanesthetized decerebrate preparation of the rat, we demonstrated rapid acute recovery of arterial pressure following C₁ transection occurring during the course of several hours (Fig. 9) (Ghali, 2019a). We accordingly believe the use of an unanesthetized decerebrate preparation of the adult rat will continue to be revelatory of critical neuronal network dynamics, not otherwise demonstrable utilizing anesthetized preparations by eschewing the confounding effects of anesthesia upon neuronal ensembles and their oscillatory synchrony (Ghali and Marchenko, 2015, 2016a,b; Ghali, 2015; Marchenko et al., 2012).

Burgeoning evidence continues to accrue precisely detailing the mechanisms by which brainstem neuronal ensembles and propriobulbar circuitry elegantly generate the respiratory rhythm and pattern, though these mechanisms have remained in controversy since the discovery of the pre-Bötzinger complex in 1991 as the kernel for inspiratory rhythm generation by the seminal serial transection studies of Smith et al. (1991) using the in vitro preparation of the neonatal rat. In vitro brainstem slices containing the preBötzinger complex consistently and reproducibly generate a regular respiratory rhythm with reduced natural sources of tonic drive (Gray et al., 2010; Morgado Valle and Beltran Parrazal, 2017), initially leading several researchers to propose fast inhibitory synaptic neurotransmission may not be critical to form the rhythm proper. Moreover, respiratory rhythmic activity evident in neurons and hypoglossal neural efferent activity of brainstem derived slice preparations containing the preBötzinger complex classically and characteristically persist in the presence of GABAergic and/or glycinergic antagonists. Studies have consequently interpreted these findings to indicate pacemaker mechanisms may be capable of independently generating respiratory rhythmic activity (Gray et al., 2010; Molkov et al., 2017; Morgado Valle et al., 2010; Smith et al., 1991). However, rhythmic phasic discharge manifest in neuronal and neural efferent output in brainstem slice preparations in vitro may arise consequent to transmembrane voltage depolarization from high concentrations of potassium used to augment natural sources of tonic drive, an assertion theoretically proposed by, and corroborated by modeling studies (Rybak et al., 2014). The use of high concentrations of potassium in vitro according represents a critical confound which invokes the necessity of utilizing in vivo studies in order to determine the necessity of fast inhibitory synaptic neurotransmission to generate triphasic eupnea characterizing the normal breathing rhythm and pattern (Marchenko et al., 2016).

Several studies have accordingly investigated breathing rhythm generation and pattern formation in anesthetized preparations in vivo. In a study conducted by Jancewzski, microinjections of the GABA_Aergic antagonist bicuculline and the glycinergic antagonist strychnine in Bötzinger and preBötzinger complexes in spontaneously breathing anesthetized rats powerfully modulated the Hering Breuer reflex in the vagal intact condition, though respiratory rhythmic activity persisted and was unaltered by microinjections of these compounds in the medulla in the vagotomized condition. The researchers interpreted the results to collectively indicate the core respiratory rhythm and pattern generating circuitry organizing eupnea may operate independently of fast inhibitory neurosynaptic transmission. Conversely, several years prior, microinjections of pharmacological agonists and antagonists of GABAergic and glycinergic neurotransmission in Bötzinger and preBötzinger complex Bongianni were demonstrated to variably perturb the respiratory rhythm and pattern, evidencing a critical role of fast inhibitory neurosynaptic transmission in generating the breathing rhythm and pattern in anesthetized rabbits in vivo.

Marchenko et al. (2016) corroboratively and commensurately demonstrated microinjections of gabazine and strychnine in preBötzinger (Fig. 25) and Bötzinger (Fig. 26) complexes significantly compromised respiratory rhythmic activity and often abolished the eupneic pattern altogether in the in situ unanesthetized preparation of the decerebrate juvenile rat and anesthetized preparation of the adult rat. Accordingly, the pacemaker currents generating intrinsic bursting behavior of preBötzinger complex characterizing pre-inspiratory, pre-inspiratory inspiratory, and decrementing early-inspiratory neurons exhibiting lower threshold for native spontaneous depolarization may prove capable of generating respiratory rhythmic activity in reduced preparations with a reduction of natural sources of tonic drive and during hypoxic conditions compromising the integrity and functionality of inhibitory network elements (Ghali, 2019c), though fast inhibitory synaptic neurotransmission and network mechanisms may be critically required in order to generate triphasic eupneic discharge during non-hypoxic normocapnic conditions at rest (Ghali, 2019c). Specifically, we believe and accordingly venture to propose microinjections of antagonists of persistent sodium (riluzole) and/or calcium activated cationic (cadmium) pacemaker currents and voltage-gated calcium channels (Ω-conotoxin) and specific pharmacological agonists and antagonists of GABAergic (e.g., gabazine) and glycinergic (e.g., strychnine) signaling in an unanesthetized preparation of the decerebrate rat will significantly and precisely elucidate the role of pacemaker currents and fast inhibitory synaptic neurotransmission in respiratory rhythmogenesis and pattern formation in the in vivo condition with the full complement network connectivity. We also seek to replicate and extend the seminal serial transection studies conducted by Smith identifying the preBötzinger complex as the principal kernel of inspiratory rhythm generation using in vitro brainstem slices of the neonatal rat to the in vivo unanesthetized decerebrate adult rat commencing at the pontomesencephalic border and progressing caudally towards and through the cervical cord in order to identify putative novel oscillators generating rhythmic activity in respiratory related neural discharges residing within the brainstem and spinal cord (Ghali and Marchenko, 2016a). We will accordingly electrophysiologically interrogate oscillator locations identified by the serial transection studies by performing individual extracellular unit recordings. This set of experiments conducted properly in an unanesthetized decerebrate preparation of the rat will contribute to elucidating types of breathing rhythmicity generable by brainstem and/or spinal cord in the presence of pharmacological antagonism of pacemaker activity and/or fast inhibitory synaptic neurotransmission and coordinately identify novel pacemakers.

Sympathetic-mediated accentuation of the arteriolar tone augments afterload and of venous tone augments right and left ventricular preload and force of myocardial contraction via Frank-Starling mechanisms, collectively increasing the magnitude of the arterial blood pressure; withdrawal of sympathetic drive generates reciprocal effects (Ghali, 2017a,b). Beyond a general consensus that supraspinal brainstem oscillators generate the resting sympathetic tone converging on maintaining arteriolar and venous tone within a physiological range and supporting sinoatrial chronotropy, atrioventricular dromotropy, and myocardial inotropy in the bulbospinal intact condition, there exists only controversy (Ghali, 2017a,b). Also, whether brainstem sympathetic oscillators principally utilize pacemaker (Sun et al., 1988) versus network (Lipski et al., 1996) mechanisms to generate coherent and correlated rhythmic activity remains in question. The concept of a critical zone residing within the brainstem generating and maintaining the resting vasomotor tone was initially proposed when Dittmar (1873) demonstrated that destruction of parts of the rostral ventrolateral medulla (RVLM) elicited precipitous declines in the arterial pressure. Investigators would later demonstrate that specific electrolytic lesioning of the RVLM reduces arterial blood pressure (ABP) to levels similar to those of observed following spinalization in anesthetized cats, rabbits, and rats Recovery of blood pressure in RVLM-lesioned rats to normal values following resuscitation from the influences of anesthesia (Cochrane and Nathan, 1989) provided evidence indicating other brainstem regions may contain presympathetic units and coordinately provide bulbospinal drive to spinal cord preganglionic sympathetic neurons and compensate for compromise of presympathetic drive provided by the RVLM.

Accordingly, one set of hypotheses proposes presympathetic neurons of the RVLM, rostral ventromedial medulla, caudal raphé, ventrolateral metencephalic tegmentum, and paraventricular nucleus generate coherent and correlated activity natively. hypothalamic and brainstem loci containing nuclearly arranged presympathetic units may receive tonic excitatory drive from other areas distributed throughout the neuraxis. The works of Barman and Gebber (Barman, 2019) and Dempesy et al. (1995) have extensively characterized the role of the medullary lateral tegmental field, a once mysterious, poorly-characterized, and ill-defined zones, in generating and modulating the activity of the rostral ventrolateral medullary presympathetic units and thus intermediolateral cell column preganglionic sympathetic neurons (Barman, 2019; Ghali, 2017a). LTF lesioning reduces arterial blood pressure and attenuates RVLM stimulation-induced pressor responses . Combined lesioning of RVLM and LTF precipitate catastrophic reductions in both ABP and heart rate. Accordingly, a few studies have astutely ventured to propose LTF may represent the originate source of basal sympathetic tone. Neurons extant throughout the expanse of the medullary division of the lateral tegmental field (LTF), an exclusively propriobulbar entity (c.f. pre-Bötzinger complex - the propriobulbar kernel of inspiratory rhythm generation), discharge prior and project to presympathetic units within the RVLM and caudal raphé and exhibit activity correlated with the cardiac-related rhythm in sympathetic nerve discharge. We interpret these data to collectively indicate the LTF originates basal sympathoexcitation. The medullary LTF also coordinates a variety of reflexes (i.e., baroreflex, Bezold-Jarisch reflex) (Barman et al., 2005; Jarisch and Henze, 1937; von Bezold and Hirt, 1867). Convergence of a panoply of descending afferents deriving from the infralimbic cortex, associated limbic structures, and vestibular inputs upon the medullary LTF suggests a critical role of this zone in the sympathetic responses to fear and coordinating the sympathetic response with emesis proper (Yates et al., 1995). The medullary LTF appears to play an indispensable and integrative role by crossmodally modulating the respiratory impulse and arterial blood pressure in response to a host of descending and peripheral stimuli. We seek to determine the effects of microinjections of pharmacological antagonists of persistent sodium and calcium activated nonselective cationic currents and pharmacological agonists and antagonists of GABAergic and glycinergic in medullary lateral tegmental field and rostral ventrolateral medulla upon the arterial pressure and sympathetic output in unanesthetized decerebrate rats.

Distinct though overlapping neuronal ensembles residing within the caudal medulla and upper cervical spinal cord contribute to generating accentuations of sympathetic activity, pressor responses, and augmentation of cerebral blood flow (Golanov et al., 2001; Goodchild and Moon, 2009; Seyedabadi et al., 2006). Medullocervical pressor area neuronal somata convey efferent axons supplying thoracolumbar intermediolateral cell column preganglionic sympathetic neurons. glutamate microstimulation of which evokes marked sympathetic pressor responses natively and in the presence of muscimol chemical inhibition of rostral ventrolateral medullary neurons in anesthetized rats (Seyedabadi et al., 2006), indicating the medullocervical pressor area (MCPA) mediates pressor responses independently of the rostral ventrolateral medulla. Vesicular glutamate transporter types 1 (VGlut1) and 2 (VGlut2) messenger ribonucleic acid (mRNA) positivity indicates a glutamatergic chemical phenotype of these units. (Goodchild and Moon, 2009; Seyedabadi et al., 2006). Activation of hypoxia sensitive rostral ventrolateral medullary reticulospinal units amplifies cerebral blood flow through the sequential activation of the medullary vasodilator area and subthalamic vasodilator area (Golanov et al., 2001). We seek to extend and further dissect the implications of these findings by conducting a similar set of investigations in unanesthetized decerebrate animals absent the confounding influences of anesthesia seeking to determine responses of cerebral blood flow, sympathetic activity, and phrenic nerve discharge to microinjections of glutamate and pharmacological agonists and antagonists of GABAergic and glycinergic signaling in in these zones. Therapeutic electrical or chemical microstimulation of the medullocervical pressor area (Goodchild and Moon, 2009; Seyedabadi et al., 2006) may be used to augment arterial pressure during the acute phase of spinal shock following spinal cord injury and of the medullary and subthalamic vasodilator zones (Golanov et al., 2001) may be used to enhance collateral blood flow to cerebral parenchyma rendered ischemic by thromboembolic or dissective large vessel occlusion, subarachnoid hemorrhage-related vasospasm, microcirculatory dysfunction, or global hypoxia from intracranial hypertension and amplifying blood flows to ischemic zones of gliomas, accordingly rendering these lesions more responsive to the cytotoxic effects of chemotherapeutic agents and stereotactic radiosurgery.

Experimental transection of the spinal cord most commonly precipitously reduces mammalian systolic arterial pressure to approximately 40 to 50 mmHg chiefly consequent to the annihilative interruption of descending brainstem presympathetic bulbospinal excitatory drive provided to preganglionic sympathetic fibers residing within the intermediolateral cell column of the thoracolumbar spinal cord. Spinal transection exerts variable effects on the arterial blood pressure and sympathetic nerve activity. Some investigators observe precipitous declines and others report only minimal decrements below normal values (Ghali, 2019a; Sherrington, 1906). Studies have accordingly reported arterial blood pressure recovers with varying kinetics. According to the reported experience of some studies, recovery of arterial blood pressure occurs within several days following transective interruption of bulbar neuronal inputs to spinal cord preganglionic sympathetic neurons. In other studies, the recovery occurs within weeks to months following spinal cord injury. We have previously demonstrated recovery of arterial pressure towards near normal and normal values following cervicomedullary transection in the unanesthetized preparation of the decerebrate rat occurring on the order of hours following injury. These studies accordingly provide evidence indicating a powerful capacity of neuronal ensembles constituting propriospinal interneuronal networks to generate sympathetic tone independently of supraspinal inputs contributing to the recovery of arterial blood pressure (Ghali, 2019a). Recovery of arterial blood pressure to near normal values following bulbospinal dissociation indicates networks residing within the myelic substance must endogenously generate coherent and correlated neuronal activity distributing to sympathetic neural efferent activity, though researchers have yet to specifically neuroanatomically and neurophysiologically characterize and interrogate these networks. Evidently, the thorough elucidation of mechanisms generating and modulating spinal sympathetic output following bulbospinal dissociation require further studies. We accordingly seek to systematically evaluate the contribution of the medullocervical pressor area and spinal sympathetic oscillators, intermediolateral cell column preganglionic sympathetic neurons, and dorsal root afferents to recovery of arterial blood pressure following cervicomedullary transection prior to and following dorsal rhizotomy in the unanesthetized decerebrate rat. We also seek to elucidate whether spinal oscillators chiefly utilize pacemaker versus network mechanisms to generate sympathetic activity. We will achieve these goals by microinjecting pharmacological agonists and antagonists of glutamatergic and GABA_Aergic and glycinergic signaling, and by downregulating identified persistent sodium and calcium activated nonselective cationic currents and/or voltage-gated calcium channels gated via microinjections of either or riluzole, cadmium, and/or Ω-conotoxin.

Studies have extensively interrogated and attempted to describe the mechanisms contributing to the generation of oscillatory variability in sympathetic neuronal, sympathetic neural efferent, and cardiovascular hemodynamic spectra. The existence of these variabilities in cardiovascular and autonomic neural outflow spectra has inspired a multitude of fruitful investigations. The sympathetic discharge contains nested fast sympathetic rhythms discharging with central tendencies approximating 10 Hz (present exclusively in unanesthetized decerebrate animals) and 2-6 Hz and coherent and correlated with spectral peaks in cardiac sympathetic nerve discharge and arterial blood pressure and much slower sympathetic rhythms (Ghali, 2017a). These slow oscillations are collectively comprised of variably defined very low ( < 0.1 Hz, varies according to study), low (~0.1-0.4 Hz, varies according to species), and high frequency (~0.25-0.4 Hz, varies according to respiratory frequency) bands of variability manifesting in the arterial pressure waveform, cardiac interval, and sympathetic and parasympathetic neuronal and neural efferent spectra. Respiratory sinus arrhythmia generates the high frequency band of sympathetic neural and hemodynamic spectral variability reflecting sinoatrial acceleration inspiratory related inhibition of cardiovagal premotoneuronal discharge. Mayer waves constitute the low frequency of variability generated by a complex interplay among and between central and peripheral mechanisms. The rhythmic contraction and relaxation of vascular smooth muscle, accordingly termed vasogenic autorhythmicity, generates very low frequency oscillations.

Studies have alternatively demonstrated abolition or persistence with spectral power reduction of Mayer wave oscillations following sinoaortic denervation mediated interruption of the baroreflex arc. Montano demonstrated the presence of Mayer waves in spectra of medullary lateral tegmental field, rostral ventrolateral medulla, caudal ventrolateral medulla, and caudal raphe neurons in unanesthetized decerebrate and pentobarbital anesthetized cats. Montano later demonstrated persistence of Mayer waves in arterial pressure following high cervical transection in unanesthetized vagotomized midcollicularly (i.e., intercollicularly; through a transverse plane bisecting the superior and inferior colliculi) decerebrate cats. We have interpreted the data and our anecdotal observations to collectively indicate Mayer waves are chiefly generated by supraspinal and spinal oscillators and are modulated and synchronized by baroreflex influences upon neuronal ensembles. We accordingly propose autochthonous oscillations of the arterioles may be mechanotransduced through the neural interstitium and generate oscillations of somatodendritic and axonal neurolemmal membranes and biophysical properties of the neurons at correspondent frequencies and at Mayer wave frequencies through the out-of-phase summation of lower frequency rhythms. We accordingly seek to evaluate the effects of baroloading, barounloading, sinoaortic denervation, chemoreceptor activation (e.g., phenylbiguanide, hypercapnia, hypoxia), pulmonary stretch receptor loading and unloading, and high cervical transection on the peak frequency, spectral band power, and coherence of Mayer wave oscillations amongst and between central sympathetic and nonsympathetic neurons, sympathetic neural efferent activity (e.g., cervical sympathetic nerve, thoracic splanchnic sympathetic nerve, renal sympathetic nerve), and arterial blood pressure. We also seek to determine coherence amongst and between neurons of supraspinal and spinal sympathetic oscillators at the Mayer wave frequency band and the attendant effects of glutamate microstimulation and muscimol chemical inhibition. brainstem zones generating Mayer wave oscillations. Our studies will accordingly determine the origins of Mayer wave genesis using carefully conducted experiments in the unanesthetized decerebrate preparation of the adult rat. The implications of this rhythmic behavior and the mechanisms of its generation are extensive and profound.

The most useful scheme considers the brainstem to be organized into discrete nuclear zones diffusely distributed throughout non-discretely organized reticular networks (Brownstone and Chopek, 2018; Marlinskii, 1992; Opris et al., 2019; Takakusaki et al., 2016; Yasargil and Sandri, 1990). Brainstem nuclei are empirically interrogable and experimentally tenable. It proves a simple matter to perform individual or multiunit recordings of, and determine the effects of microinjections of pharmacological agonists and antagonists of different neurochemical signaling pathways upon, neurons in these zones (Marchenko et al., 2016). The brainstem nuclei are visually demonstrable in histologic specimens, discretely bounded by a variable transition zone from reticularly or loosely-organized cells (Dobbins and Feldman, 1994). Consequently, the majority of neurophysiologists seeking to illuminatively interrogate the mechanistic underpinnings of network interactions amongst neurons of the bulb, and those in hopes of generating a successful set of experiments sustaining a continuous influx of government or research foundation issued grant funding, preferentially subject discretely arranged nuclear zones to experimental interrogation. The role of reticular zones in modulating central pattern generation of breathing, sympathetic oscillations, and locomotor discharge, escaping the precision of certainty, remains largely unexplored. We may parsimoniously conceive of the brainstem reticulospinal network to be organized in nodal architecture, with nodes variably or alternatively excitatory or inhibitory. Brainstem reticulospinal units convey excitatory and inhibitory modulatory synaptic drives shaping phasic and tonic discharge of motoneurons supplying upper and lower extremity and thoracic, abdominal, and pelvic musculature. Accordingly, a close and considerate evaluation of the literature readily reveals this most mystical of Nature’s creations to tacitly conceal the mechanisms by which to effectively alleviate a substantial fraction of the most severe and catastrophic of the human morbidity burden ensuing from ischemia to the cerebrum.

We have extensively characterized the architectural organization of the brainstem reticular formation and generally determined the course, laterality, and neurochemical character of reticulospinal tracts and pathways conveying excitatory and inhibitory synaptic drives modulating phasic and tonic activities of motoneurons (Brownstone and Chopek, 2018). Approximately 85% of reticulospinal axons extant within the substance of the bulb coordinately provide synaptic drives to motoneurons of the cervical and lumbar enlargements. Cervically projecting brainstem reticulospinal units often provide both ipsilateral and contralateral synaptic drives to spinal pre-motoneuronal interneurons and motoneurons. The pedunculopontine (PPN) and cuneiform (CnF) nuclei collectively comprise the mesencephalic locomotor region of the mesencephalic reticular formation (Opris et al., 2019). These nuclei variably and specifically project to zones containing heterogeneously semi-compactly organized neuronal ensembles constituting the medullary reticular formation (the experimental stimulation of which may independently generate locomotion). Target efferents of the mesencephalic locomotor region include the nucleus gigantocellularis (CnF, PPN), nucleus gigantocellularis pars alpha (CnF, PPN), nucleus gigantocellularis pars ventralis (CnF, PPN), lateral nucleus paragigantocellularis (CnF, PPN), dorsal paragigantocellularis (PPN), the caudal extension of the nucleus gigantocellularis (PPN), and the ventral division of the caudal medial medullary reticular formation (PPN). The metencephalic reticular formation may be conceptually divided into a medial zone, comprised of the nucleus reticularis pontis oralis rostrally and the nucleus reticularis pontis caudalis caudally, the neurons of which project to lateral spinal cord motoneurons innervating appendicular hindlimb musculature and a lateral zone, the neurons of which project to medially located spinal cord motoneurons innervating the axial muscles supporting posture. The medial metencephalic reticular formation extends caudally into the medullary reticular formation dorsal to the nucleus gigantocellularis. The dorsally related pontine tegmental field may be conceptually divided into ventral, dorsal, and lateral zones. Pontine reticulospinal units span the rostrocaudal extent of the metencephalon, with neurons projecting ipsilaterally in greater abundance compared with those projecting contralaterally. Contralaterally projecting reticulospinal units are preferentially located within the caudal pons (Sivertsen et al., 2016).

Median, medial, and lateral divisions constitute the medullary reticular formation. Median midline raphe serotonergic reticulospinal neurons facilitate spinal motoneuronal discharge (Brownstone and Chopek, 2018). The nucleus gigantocellularis, nucleus gigantocellularis pars alpha, and nucleus gigantocellularis ventralis, the caudal extension of which may be divided into ventral and dorsal components, effectively constitute the medial medullary reticular formation (Sivertsen et al., 2016). The lateral paragigantocellularis, dorsal paragigantocellularis, and the dorsolateral parvocellular reticular formation collectively constitute the lateral medullary reticular formation. The intermediate division of the medullary reticular formation may be found interposed between the nucleus gigantocellularis ventrally and the dorsolateral parvocellular reticular formation and the ventral and dorsal divisions of the caudal extension of the nucleus gigantocellularis dorsally. Medullary reticulospinal neuronal somatodendritic membranes receive excitatory synaptic drive from axons of neurons of the contralateral cerebellar nuclei and the ipsilateral subthalamic locomotor region. The axons of medullary reticulospinal units course within the ventrolateral funicular division of the myelic substance en route to making synaptic contacts with ipsilateral or contralateral neurolemmal somatodendritic membranes of spinal cord motoneurons and pre-motoneuronal commissural interneurons circumscribing the central spinal canal providing inhibitory synaptic drive to ventral horn motoneurons. [Nota Bene: The majority of spinal cord commissural interneurons expressing the 65 and 67 kilodalton isoforms of glutamate decarboxylase and thus utilize GABAergic fast inhibitory neurotransmission, and likely represent a critical mechanism providing inhibitory modulation of patterned motor output (Yasargil and Sandri, 1990)]. Experimental stimulation of stereotactically identified zones corresponding with brainstem reticulospinal neurons alternatively generates excitatory, inhibitory, or biphasic postsynaptic potentials of pre-motoneuronal interneurons and motoneurons innervating flexor or extensor skeletal myocytes of the head, face, neck, and upper and lower extremities via ipsilateral and/or contralateral monosynaptic and/or polysynaptic relays (Brownstone and Chopek, 2018) and may generate movements across multiple joints. The firing of reticulospinal neurons with somata extant within the medial medullary reticular formation drives, and modulates endogenous spinally generated, patterned locomotor discharge (Marlinskii, 1992), though recruitment of lateral paragigantocellularis reticulospinal neurons may prove requisite in order to generate higher velocity locomotor activity. A proportion of gigantocellularis nucleus reticulospinal neurons convey an inhibitory modulation via glycinergic signaling upon target efferent interneurons and motoneurons.

We have thus provided a cursory, though concise and pointed discourse discussing the general architectural organization of brainstem reticulospinal tracts and pathways conveying excitatory and inhibitory synaptic drives modulating phasic and tonic activities of motoneurons. We accordingly propose studies employing retrograde transsynaptic labeling (e.g., using pseudorabies virus) techniques will thoroughly characterize the architectonic organization of the brainstem reticulospinal network (see Dobbins and Feldman (1994); Lane et al. (2008, 2009a,b)). The neurochemical identity of brainstem reticulospinal units may be determined by immunohistochemically labeling transverse slices sectioned from brainstem specimens using secondary fluorescent anti-immunoglobulin (amplification technique) targeting primary non-fluorescent anti-enzyme (e.g., 65 and 67 kilodalton isoforms of glutamate decarboxylase [GAD65/67]) or anti-cell surface transmembrane protein (e.g., glycine transporter type 2 [GLYT2]) antibody (molecular identification technique) (see Marchenko et al. (2015)). Stimulation of brainstem zones corresponding with, and containing an abundance of, spinally projecting reticular neurons possessing a GABAergic or glycinergic phenotype (e.g., medullary nucleus gigantocellularis, gigantocellular depressor area) coordinately performed with electrophysiological recordings of individual motoneuronal units, motoneuronal population activity (motor nucleus multiunit records or neurogram), or muscle discharge in unanesthetized decerebrate preparations will thoroughly characterize the electrophysiological behavior of the bulboreticulospinal network and thus characterize the nature of bulboreticular modulation of the brainstem and spinal motoneurons (Brownstone and Chopek, 2018). Intracellular neuronal recordings will determine locoregional heterogeneity of response patterns and dynamics of excitatory and inhibitory postsynaptic potentials in response to electrical or chemical microstimulation of zones disparately distributed throughout the brainstem reticular network.

We venture to propose reduced brainstem reticulospinal GABAergic and glycinergic synaptic drives conveyed to brainstem and spinal motor networks attenuates fast synchronous oscillatory coherence amongst, and signal to noise ratio between phasic and tonic discharge of, motoneurons, representing the principal pathogenic mechanisms generating spastic paresis (see Marchenko et al., 2015). Accordingly, determining the amplitude of, and ratio between, phasic and tonic components of individual and population motoneuronal activity and elucidating properties and spatiotemporal dynamics of the responses of phasic and tonic components of motoneuronal discharge to electrical or chemical microstimulation or inhibition of nodes disparately distributed throughout the brainstem reticular network individually or in unison in the setting of various motor disorders (compared with the normal physiological condition) will enhance our capacity to more precisely interrogate the specific neurobiological mechanisms giving rise to neurogenic muscle spasticity. Selectively augmenting GABAergic and glycinergic synaptic drive to motoneurons via electrical or chemical microstimulation of brainstem reticular network zones generates effects electrophysiologically distinct from parenteral administration of a pharmacological GABAergic agonist (e.g., baclofen, benzodiazepines, cyclobenzaprine), the current medical standard of care in the treatment of spasticity, which will coordinately augment phasic and tonic inhibitory drive to excitatory and inhibitory interneurons. While medical spasmolytics will certainly reduce the tonic background, activity generated by motoneurons, the use of these agents may coordinately excessively reduce the phasic discharge of motoneurons and generate sedative effects reducing the wakeful period and motivation to move. We suggest neural efferent outputs innervating limb and torso musculature may represent standardized metrics of evaluating power and smoothness of movement and postural output. Well-conducted, these studies should expectedly contribute to evolving an experimentally interrogable conceptualization of brainstem reticular modulation of motor output coordinately with developing and forming an understanding of the nature and character of supraspinal modulatory influences upon brainstem and spinal motor neuronal ensembles deriving from neural network arrays of the deep basal nuclei, supplementary, premotor, and motor cortices, cerebellar cortex and deep nuclei, ventral anterior, ventral thalamic, and centromedian nuclei, and brainstem zones in health and disease. These findings will collectively guide the rational development of therapies aimed at ameliorating motor disorders and shape our understanding of the benefit expectedly derived from these strategies.

We accordingly propose the following set of empirically evaluable therapeutic strategies designed to judiciously modify the firing behavior of bulbar reticulospinal units predicated and theoretically premised upon the described framework and presented conceptualization of brainstem reticular network structure and function may enhance and optimize the power, smoothness, and coordination of muscle contractions in individuals suffering from various motor disorders (see Marchenko et al. (2015)). Architectonic and electrophysiological characterization of GABAergic and glycinergic inhibitory reticulospinal neurons will effectively identify candidate zones which may be therapeutically stimulated in order to liberate smooth fluidity of skeletal muscle movement through the diminution of interregnum background erratic tonic activity interposed between purposeful contractions (Brownstone and Chopek, 2018). Augmenting the activity of these cells may effectively enhance resolution and ratio between phasic and tonic components of motoneuronal activity by amplifying coherent oscillatory synchrony amongst and between the individual motoneurons and by conveying a descending inhibitory modulation upon hyperactive spinal myotatic stretch reflex arcs. Stimulation of zones corresponding with nucleus gigantocellularis glycinergic units will provide a parallel inhibitory modulation of spinal motoneurons synergistic with the activity of Renshaw cells. Attenuating tonic background activity of brainstem and spinal motoneurons will amplify cellular responsivity to descending phasic synaptic drives by reducing the haphazard metabolic consumption of high energy phosphates (creatine phosphate) and phospho-phosphate esters (e.g., adenosine triphosphate, guanosine triphosphate) by uncoordinated fasciculations of skeletal muscle diminutively generative of appreciable displacements of the limb in space and of work against the gravitational force.

Neurosurgeons have extensively and variably utilized transcranial magnetic and deep brain stimulation of the motor cortex (Pagni et al., 2008), internal capsule (Franzini et al., 2008), thalamus (Yang et al., 2020), globus pallidus internus (Tan et al., 2016), subthalamic nucleus (Lin et al., 2020a; Tan et al., 2016; Vassal et al., 2019), pedunculopontine nucleus (Lin et al., 2020b), and cerebellum (Bologna and Beradrelli, 2018; Franҫa et al., 2018; Okromelidze et al., 2020; Rezaee et al., 2020) to ameliorate Parkinsonian tremor, rigidity, and bradykinesia (Lin et al., 2020a; Pagni et al., 2008; Vassal et al., 2019; Tan et al., 2016), essential tremor (Blaabjerg et al., 2020), and dystonia (Okromelidze et al., 2020; Pagni et al., 2008). Studies suggest, by extrapolative deduction, the putative efficacy of such techniques in relieving spasticity (Franzini et al., 2008; Pagni et al., 2008; Rezaee et al., 2020; Vassal et al., 2019). We may therapeutically enhance and amplify the activity and discharge of brainstem GABAergic and glycinergic reticulospinal neurons in human patients through electrical stimulation or by administering pharmacological agonists of specific receptor subtypes parenterally, epidurally, intraventricularly (via a surgically placed Ommaya reservoir), or locally microiontophoretically. High frequency electrical stimulation of brainstem zones containing GABAergic or glycinergic reticulospinal units or regions conveying excitatory modulatory inputs to these regions via transcranial magnetic or deep brain stimulation could effectively augment brainstem inhibitory drive conveyed to brainstem and spinal motor networks. A modest reduction in tonic background motoneuronal activity may significantly enhance motor efficiency Local implantation of neural stem cells in specific zones of the brainstem reticular network in the presence of neuronal growth factors driving differentiation towards a neuronal lineage may therapeutically and specifically repopulate brainstem reticular regions with GABAergic and glycinergic reticulospinal neurons conveying descending inhibitory synaptic drives to brainstem and spinal motoneurons. In the setting of brainstem reticular neural stem cell implantation, high frequency stimulation may be synergistically used to promote local cell specific neurogenesis and amplify the extent of distal axodendritic synapse formation with motoneurons and interneurons constituting brainstem and spinal motor networks and improve electrochemical neural transduction efficiency. Conducting terminal neurophysiological experiments on unanesthetized decerebrate animals will permit the precise interrogation of the specific effects of electrically or chemically microstimulating brainstem zones corresponding with the location of GABAergic and glycinergic reticulospinal neurons and the complement of empirical therapeutic strategies upon the phasic and tonic components of motoneuronal discharge absent the confounding influences of anesthetic mediated potentiation of fast inhibitory synaptic neurotransmission in acute and chronic models of spasticity.

Classic microsurgical approaches may facilitate the placement of stimulatory electrodes or microcatheters designed to deliver therapeutic compounds within brainstem zones corresponding with the location of GABAergic and glycinergic reticulospinal neurons (Rhoton et al., 2000a,b; Spetzler et al., 2015). Microsurgical exposure of the brainstem ventrally may be achieved via transoral transpharyngeal transclival or transmaxillary routes, ventrolaterally via suboccipital trephination, C₁ laminectomy, and far lateral approach, and dorsally via suboccipital craniectomy with transvermian or transtelovelar approach in order to gain access to therapeutically modifiable brainstem reticulospinal neurons (Rhoton et al., 2000a,b; Spetzler et al., 2015). We accordingly indicate the critical need to generate electrophysiological and microiontophoretic dose response curves in order to establish threshold and lowest effect levels, optimal stimulation parameters, and toxicity levels in preclinical animal models and human patients. High frequency stimulation or glutamate microstimulation of suprabulbar and rhomebencephalic zones providing synaptic drives to reticulospinal units may generate NMDA dependent synaptic plasticity and long term synaptic potentiation (Kandel, 1979; Kandel et al., 2000); lower frequencies of stimulation could inadvertently cause long term synaptic depression and reduce the efficiency and fidelity of neurosynaptic conduction (Kandel, 1979; Kandel et al., 2000). Accordingly, differential kinetics of cytosolic calcium rates of rise represent the principal determinant of whether a postsynaptic dendrite experiences enhancement or attenuation of neurosynaptic transduction efficiency. The described set of strategies may be therapeutically exploited in order to enhance the activity of brainstem reticulospinal cells and indirectly excite a plethora of spinal commissural GABAergic and glycinergic interneurons conveying inhibitory modulation of contralateral ventral horn motoneuronal transmembrane potential.