Autoradiographic mapping of cerebral blood flow responses to cholinergic stimulation of the rat substantia innominata: modulatory effect of galanin

In order to analyze the precise cerebrovascular effects of a specific cholinergic stimulation of the rat substantia innominata and their modulation by galanin, cerebral blood flow was measured by the [¹⁴C]-iodoantipyrine autoradiographic method in anesthetized (urethane and α-chloralose), artificially ventilated male Sprague–Dawley rats that received a microinjection into the substantia innominata of saline (n=7), or 63 pmol of galanin (n=8), or 50 nmoles of carbachol (n=6) or a coinjection of carbachol and galanin (n=8). Significant carbachol-induced cerebral blood flow increases were noted in ipsilateral cortices (+36%, p<0.01 in the cingulate to +82%, p<0.01 in the parietal somatosensory cortices), but also in ipsilateral hippocampus and ipsilateral thalamus. These cerebral blood flow increases were abolished by the coinjection of carbachol and galanin, while infusions of galanin alone failed to affect cerebral blood flow. Cholinergic stimulation of the substantia innominata represents thus a good model for the analysis of the detailed pharmacological properties of the cholinergic vasodilatatory basalocortical system. The existence of an inhibitory galaninergic modulation of this system could be of particular interest, in terms of cerebrovascular reactivity, in various neurodegenerative states.     

Neurochemical stimulation of the rat substantia innominata increases cerebral blood flow (but not glucose use) through the parallel activation of cholinergic and non-cholinergic pathways

Neurochemical activation of the substantia innominata (SI) in the rat, through the direct injection of the cholinergic agonist carbachol, has been reported to induce large increases in cerebral blood flow (CBF) throughout cortical and subcortical projection regions. The present study aimed to determine whether the vasomotor responses to cholinergic stimulation of the SI were, or were not, the consequence of an increase in metabolic activity. To this end, coupled measurements of CBF and cerebral glucose use (CGU) were undertaken during carbachol-elicited stimulation of the SI. Infusion of carbachol into the basal forebrain induced significant CBF increases in several ipsilateral cortical and subcortical areas including the amygdala. In contrast, CGU increased only in the ipsilateral amygdala and SI. Thus, we tested the hypothesis of a direct neurogenic, rather than metabolic, contribution of the basalocortical system. In this respect, carbachol-elicited stimulation resulted in significant increases in extracellular acetylcholine concentrations in the ipsilateral parietal cortex; systemic pretreatment with the muscarinic receptor antagonist scopolamine completely abolished the increase in cortical CBF elicited by cholinergic stimulation of the SI in the ipsilateral frontoparietal motor cortex while it failed to affect the increase observed in the ipsilateral temporal cortex. Several conclusions can be drawn from the present study. The stimulation of the SI by carbachol induces an increase in CBF that can be dissociated from changes in underlying glucose metabolism. Secondly, these induced changes in cortical CBF are paralleled by an increase in acetylcholine release. Lastly, the failure of scopolamine to block the flow response in all cortical regions would suggest that SI stimulation will evoke the release of vasodilatatory neurotransmitter(s) as well as acetylcholine itself.     

Local cholinergic mechanisms participate in the increase in cortical cerebral blood flow elicited by electrical stimulation of the fastigial nucleus in rat

We sought to determine whether the increase in regional cerebral blood flow (rCBF) elicited within the cerebral cortex (CX) by electrical stimulation of the fastigial nucleus (FN) of the cerebellum is: prevented by local application of the muscarinic cholinergic receptor antagonist, atropine and temporally correlated with a stimulus-locked release of acetylcholine (ACh) from the cortical surface. Rats were anesthetized, paralyzed, ventilated, with arterial blood gases controlled and arterial pressure maintained within the autoregulated range. Bilateral craniotomies were performed over a standardized region of the sensory motor CX and superfusion devices stereotaxically positioned on the cortical surface. Cortical surface temperature, as well as pH, pCO2 and pO2 of the solutions applied to the cortex were also carefully controlled. rCBF was measured in dissected regions of frontal (FCX), parietal (PCX), and occipital cortices (OCX), caudate nucleus (CN), and hippocampus (HIPP) by the Kety principle using [14C]iodoantipyrine as indicator. Resting rCBF (ml/100 g/min) in unoperated control animals ranged from 70 +/- 5 in HIPP to 95 +/- 7 in PCX and was unaffected by bilateral craniotomies and placement of superfusion devices containing Kreb's bicarbonate buffer (vehicle) on the cortical surface. Local application of atropine (ATR, 100 microM) to the right PCX via the superfusion device did not affect resting rCBF. With FN stimulation rCBF increased bilaterally and symmetrically in all areas up to 227% in PCX. ATR application attenuated by 59% the FN-elicited increase in rCBF on the ipsilateral frontoparietal CX, without affecting blood flow in adjacent structures. ATR did not affect cortical cerebrovasodilation produced by hypercarbia (arterial pCO2 = 59.0 +/- 1.4 mm Hg). FN-stimulation resulted in a small (22%) but significant (P less than 0.05, n = 9) reduction in the release of [3H]ACh from the cortical surface, while supramaximal depolarization with 55 mM K+ increased [3H]ACh release by 251%. These studies indicate that: increases in cortical rCBF elicited by FN stimulation, but not hypercarbia, are in large part mediated by local muscarinic cholinergic receptors; resting rCBF is not tonically affected by muscarinic receptor activation; and the release of ACh from the cortical surface is, in general, reduced during FN-stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Role of nucleus basalis in cholinergic control of cortical blood flow.

The present investigation was designed to determine the effect of lesions localized to the nucleus basalis/substantia innominata (NB) on resting and cholinergically activated regional cerebral cortical blood flow (rCBF). Ibotenic acid (10 micrograms) was infused locally at 1 mm caudal to bregma, 3 mm lateral to the midline, and 8 mm below the cortical surface. Effectiveness of lesions was demonstrated by histological verification of lesion sites and determination of choline acetyltransferase activity in cerebral cortex homogenates. rCBF was measured with the autoradiographic iodo-14C-antipyrine technique. Resting rCBF was similar in the hemisphere that received the NB lesion and in the contralateral (intact) side in all regions examined. Physostigmine intravenous infusion (3.3 micrograms.kg-1.min-1) enhanced rCBF in frontal, parietal, occipital, and temporal cortex. The increase was symmetrical, however, indicating inability of NB lesion to affect this phenomenon. It is concluded that the cortical cholinergic afferents originating in the NB are not involved in the control of rCBF.     

Widespread reductions in cerebral blood flow and metabolism elicited by electrical stimulation of the parabrachial nucleus in rat

We have studied the effect of electrical stimulation of the parabrachial nucleus (PBN) and adjacent areas of dorsal pons on regional cerebral blood flow (rCBF) and glucose utilization (rCGU) in anesthetized (chloralose), paralyzed (tubocurarine) rats. rCBF and rCGU were measured in dissected tissue samples of 9 brain regions by the [¹⁴C]iodoantipyrine and [¹⁴C]2-deoxyglucose method, respectively. Electrical stimulation restricted to the medial parabrachial nucleus (PBNm, n=5) elicited significant (P < 0.05) reductions in rCBF in 7 out of 9 brain regions. Reductions were greatest in cerebral cortex (up to 35% in occipital cortex) and least in the white matter of the corpus callosum (23%). The effect on rCBF persisted after transection of the cervical sympathetic trunk (n=5). In contrast, stimulation of the lateral portion of PBN (n=5), periventricular gray (n=5) and interestingly, the nucleus locus coeruleus (n=5) failed to elicit similar changes in rCBF. PBNm stimulation also elicited decreases in rCGU (n=4) in 5 out of 9 brain areas, most notably regions of cerebral cortex. The decreases in rCGU (ΔrCGU) were linearly related to the decreases in rCBF (ΔrCBF) according to the equationΔrCBF=2.37 ΔrCGU+2.1 (r=0.72; P<0.001). We conclude that excitation of neural pathways originating in, or passing through, PBNm elicits a widespread reduction in cerebral metabolism and secondarily in blood flow (secondary vasoconstriction). Since projections of the PBNm do not involve the entire cortex, it seems likely that the effect is mediated via inhibition of diffuse cortical projections through a subcortical site.     

Deoxyglucose uptake and choline acetyltransferase activity in cerebral cortex following lesions of the nucleus basalis magnocellularis

The uptake of [3H]2-deoxyglucose (2-DG) into various brain regions of rats with unilateral or bilateral lesions of the nucleus basalis magnocellularis (nBM) was measured. The activity of choline acetyltransferase (ChAT) in these brain regions was also determined. Lesions of the nBM caused a significant decrease in cortical ChAT activity but had no effect on 2-DG accumulation. Pentobarbital treatment reduced 2-DG accumulation in all brain areas examined and these reductions were not influenced by the nBM lesions. The results indicate that a decrease in the cholinergic innervation of the cortex does not influence cortical glucose utilization. It appears unlikely, therefore, that the reported decrease in cortical glucose utilization in Alzheimer's disease is related to degeneration of the nBM-cortical cholinergic projection.     

Recovery of neocortical choline acetyltransferase activity following ibotenic acid injection into the nucleus basalis of Meynert in rats

The present experiment was designed to investigate the depletion and possible recovery of neocortical choline acetyltransferase (ChAT) levels following damage to the basal forebrain cholinergic (Ch) system in rats. A unilateral injection of ibotenic acid was made into the left nucleus basalis of Meynert (nbM). Seven days after the injection, neocortical ChAT levels had decreased 60%. After 3 months it had returned to normal. Thus this system shows extensive plasticity and recovery of function with time.     

Short-term longitudinal evaluation of cerebral blood flow in mild Alzheimer's disease

The aim of this study was to estabilish whether subtle changes in regional cerebral blood flow (rCBF) can be detected in patients with mild Alzheimer's disease (AD) over a period as short as three months. Aprospective evaluation of rCBF changes with HMPAO SPECT at baseline and after 3 and 6 months was carried out. Standard clinical dementia tests were performed in parallel. The study enrolled 13 patients with mild probable AD from an outpatient memory clinic. SPECT data collected at baseline and after three months were compared using Statistical Parametric Mapping (SPM). Significant clusters of rCBF decrement in the medial part of the temporal lobe in both hemispheres, in the orbital part of the right frontal lobe and in the inferior part of the right parietal lobe were observed. No significant differences were found using a region of interest (ROI) analysis. After six months SPM analysis showed larger clusters of significant decrement in the same areas. ROI analysis was now sensitive to the rCBF changes and significant differences were shown in all brain regions except the temporal lobes. SPM analysis of SPECT data can detect significant changes in rCBF in mild AD over a short time interval. This method might be of potential advantage for the early diagnosis of AD and for its differentiation from stable cognitive impairments. Received: 30 November 2002 / Accepted in revised form: 28 January 2003 Correspondence to: A. Venneri     

Therapeutic high-frequency stimulation of the subthalamic nucleus in Parkinson's disease produces global increases in cerebral blood flow

Chronic, high-frequency electrical stimulation of the subthalamic nuclei (STNs) has become an effective and widely used therapy in Parkinson's disease (PD), but the therapeutic mechanism is not understood. Stimulation of the STN is believed to reorganize neurophysiological activity patterns within the basal ganglia, whereas local field effects extending to tracts adjacent to the STN are viewed as sources of nontherapeutic side effects. This study is part of a larger project investigating the effects of STN stimulation on speech and regional cerebral blood flow (CBF) in human subjects with PD. While generating measures of global CBF (gCBF) to normalize regional CBF values for a subsequent combined analysis of regional CBF and speech data, we observed a third effect of this therapy: a gCBF increase. This effect was present across three estimates of gCBF ranging from values based on the highest activity voxels to those based on all voxels. The magnitude of the gCBF increase was related to the subject's duration of PD. It is not clear whether this CBF effect has a therapeutic role, but the impact of deep brain stimulation on cerebrovascular control warrants study from neuroscience, pathophysiological, and therapeutic perspectives.     

Regulation of regional cerebral blood flow by cholinergic fibers originating in the basal forebrain

The intracranial neural vasodilative system of cholinergic fibers projecting from the basal forebrain to the cortex was discovered by Biesold, Inanami, Sato and Sato (Biesold, D., Inanami, O., Sato, A., Sato, Y., 1989. Stimulation of the nucleus basalis of Meynert increases cerebral cortical blood flow in rats. Neurosci. Lett. 98, 39-44) using laser Doppler flowmetry in anesthetized rats. This cholinergic vasodilative system, which operates by increasing extracellular ACh release, relies upon activation of both muscarinic and nicotinic cholinergic receptors in the parenchyma of the cortex. Further, the involvement of nitric oxide in this cholinergic vasodilation, indicates the necessity to this system of neurons, which contain nitric oxide synthase. The increase in cortical blood flow elicited by this cholinergic vasodilative system is independent of systemic blood pressure and is not coupled to cortical metabolic rates. This cholinergic vasodilative system may be activated by somatic afferent stimulation. Most of the data presented here were obtained in anesthetized animals.     

Global reduction in cerebral blood flow and metabolism elicited from intrinsic neurons of fastigial nucleus

We sought to determine whether the global increase in regional cerebral blood flow (rCBF) produced by electrical stimulation of the rostral cerebellar fastigial nucleus (FN) is a consequence of excitation of intrinsic neurons of the FN or of axons of fibers passing through or projecting into it. Studies were conducted in rats anesthetized with chloralose, paralyzed and ventilated. rCBF was measured with [¹⁴C]iodoantipyrine as tracer and regional cerebral glucose utilization (rCGU) by [¹⁴C]2-deoxyglucose in homogenates of 11 brain regions. Neuronal perikarya in FN were excited chemically by local microinjection of the glutamate analogue kainic acid (KA) (5 nmol in 100 nl). KA elicited a transient and significant fall of arterial pressure and heart rate, the fastigial depressor response (FDR). Associated was a significant and symmetrical reduction in rCBF, to 44% of control in all regions except medulla. The response was site- and agent-specific and unrelated to the hypotension. KA also significantly and proportionally reduced, to 52% of control, rCGU in the same 10 areas of brain. In all regions the magnitudes of the reductions in rCBF and rCGU elicited by KA were linearly related. Intrinsic neurons of FN were chronically destroyed by local microinjection of the excitotoxin ibotenic acid (IBO) (10 μg/μl in 0.4 μl). Destruction of intrinsic FN neurons had no effect on resting rCBF nor on the global cerebrovascular vasodilation elicited by electrical stimulation of the FN. We conclude that: (a) excitation of intrinsic neurons of FN elicits a widespread reduction of cerebral metabolism and, secondarily, blood flow; (b) FN neurons do not exert a long-term tonic influence on brain blood flow nor metabolism; (c) the global increase in rCBF elicited by electrical stimulation of the FN is a consequence of excitation of axons projecting into or through the nucleus.     

Muscarinic cholinergic receptors mediate the cerebrovasodilation elicited by stimulation of the cerebellar fastigial nucleus in rat

We have investigated the effects of systemic administration of atropine sulfate on the global cerebrovascular vasodilation elicited by electrical stimulation of the cerebellar fastigial nucleus (FN) in chloralose-anesthetized rat. Atropine (0.3 mg/kg, i.v.) abolished the cerebrovasodilation elicited from FN but did not affect the concomitant elevation in arterial pressure and the EEG changes. We conclude that the cerebrovascular effect of FN stimulation, but not the peripheral cardiovascular or EEG changes, are mediated by cholinergic muscarinic receptors associated with cerebral vessels and/or intrinsic neural pathways.     

Effect of drinking on angiotensin-II-induced shifts in regional cerebral blood flow in the rat

A map of brain regions affected by central administration of the octapeptide angiotensin II (AII) and that would further reflect the consequences of AII's well-known dipsogenic action was developed. Regional cerebral blood flow (rCBF) as an indicator of neuronal activity was measured in conscious rats shortly after an ICV bolus injection of 100 ng AII or saline vehicle (VEH). AII-treated animals were further subdivided into two groups that were either permitted to drink [AII (W+)] or whose water was removed when drinking was attempted [AII (W-)]. When compared to VEH condition, blood flow increased significantly within 1 min after AII treatment in 33 of 53 regions sampled in those rats not given an opportunity to drink. In 11 of these 33 regions, ingestion of a small amount of water was associated with a reversal of AII-induced elevation in blood flow [i.e., AII (W+) less than AII (W-)]; these regions included the organum vasculosum lamina terminalis, rostral lateral hypothalamus, supraoptic nuclei, rostral zona incerta, and median eminence. A group of similarly treated rats exhibited a significant elevation of mean arterial blood pressure following AII treatment without significant shifts in arterial blood gases, pH, or bicarbonate. These data are consistent with prominent involvement of the anteroventral third ventricular region of the rat brain. The results further indicate that rCBF may be a sensitive measure for the identification of central sites of action of AII as a dipsogenic agent and may reveal distinctions between regions associated primarily with initiation of drinking and those reflecting the results of subsequent behavioral events.     

Deep brain stimulation of the nucleus basalis of Meynert attenuates early EEG components associated with defective sensory gating in patients with Alzheimer disease – a two‐case study

Alzheimer′s disease (AD) is associated with deterioration of memory and cognitive function and a degeneration of neurons of the nucleus basalis of Meynert (NBM). The NBM is the major input source of acetylcholine (ACh) to the cortex. The decreasing cholinergic innervation of the cortex due to degeneration of the NBM might be the cause of loss of memory function. NBM‐Deep brain stimulation (NBM‐DBS) is considered to serve as a potential therapeutic option for patients with AD by supporting residual cholinergic transmission to stabilize oscillatory activity in memory‐relevant circuits. However, whether DBS could improve sensory memory functions in patients with AD is not clear. Here, in a passive auditory oddball paradigm, patients with AD (N = 2) listened to repetitive background tones (standard tones) randomly interrupted by frequency deviants in two blocks with NBM‐DBS OFF and then NBM‐DBS ON, while age‐matched healthy controls (N = 6) repeated the experiment twice. The mismatch negativity in NBM‐DBS OFF significantly differed from controls in both blocks, but not under NBM‐DBS, which was likely due to a pronounced P50 increase overlapping with the N1 in NBM‐DBS OFF. This early complex of EEG components recovered under stimulation to a normal level as defined by responses in controls. In this temporal interval, we found in patients with NBM‐DBS ON (but not with NBM‐DBS OFF) and in controls a strong repetition suppression effect to standard tones – with more attenuated responses to frequently repeated standard tones. This highlights the role of NBM‐DBS for sensory gating of familiar auditory information into sensory memory.     

Clinical and cerebral activity changes induced by subthalamic nucleus stimulation in advanced Parkinson's disease: a prospective case-control study

Background

High-frequency stimulation of the subthalamic nucleus (STN-DBS) improves motor symptoms in advanced Parkinson's disease (PD), but the mechanisms are still unclear. Functional imaging evidenced pathological overactivity in motor cortical areas in advanced PD that can be normalized by effective therapies.

Patients and methods

We studied resting state cerebral blood flow pre-operatively and 12 months after surgery in 40 patients with advanced PD using ECD-SPECT. SPECT scans were also acquired 1 year apart in 21 matched PD controls who did not undergo surgery. Statistical analysis was performed using statistical parametric mapping (SPM2) software. In addition, we correlated brain perfusion changes after surgery with clinical improvement, assessed using the unified PD rating scale motor score (UPDRS-III).

Results

Patients showed marked motor improvement and medication reduction after surgery. Stimulated PD patients revealed bilateral rCBF decrements in motor cortical areas and prefrontal cortex bilaterally compared to pre-surgical condition as well as versus PD controls (p < .01 FDR corrected). Perfusion increases were found in cerebellum, temporal and occipital lobes. Clinical improvement was associated with perfusion decrements in primary motor and premotor cortices.

Conclusions

Effective STN-DBS is associated with neuronal activity changes in brain regions implicated in movement programming and performance. We hypothesize that clinical benefit might be associated with stimulation-induced normalization of the abnormal overactivity within the cortico-basal ganglia-thalamo-cortical motor loop in advanced PD.     

Longitudinal study of cerebral blood flow in Alzheimer's disease using single photon emission tomography

Ten patients with probable Alzheimer's disease were assessed at baseline and a mean 2 years later using a battery of neuropsychological tests, CT scans and Tc99m-HMPAO SPECT scans. The subjects had declined significantly in their functional indices. Cerebral perfusion measures declined in the parietal lobes, left hemisphere and whole brain, but the overall decline did not reach statistical significance. The decline in brain perfusion did not correlate significantly with the decline in various indices of neuropsychological function, either globally or for specific brain regions. The index of cerebral perfusion correlated significantly with global indices of neuropsychological function at baseline but not at follow-up. No single perfusion index was a significant predictor of clinical progression of dementia.     

Decrease of somatostatin receptor binding in the rat cerebral cortex after ibotenic acid lesion of the nucleus basalis magnocellularis: a quantitative autoradiographic study

The specific binding of¹²⁵I-Tyr¹¹-somatostatin-14 (¹²⁵I-Tyr¹¹-SS-14) was measured in different cortical regions after unilateral ibotenic acid lesion of the rat nucleus basalis magnocellularis (NBM). A marked loss of acetylcholinesterase-positive fibers was observed in the frontal, parietal, temporal and occipital cortices ipsilateral to the lesion. The loss of cholinergic cell bodies in the NBM was further investigated with cholineacetyltransferase (ChAT) immunohistochemistry which indeed demonstrated a loss of ChAT-positive magnocellular perikarya. Autoradiographic analyses of specific binding of¹²⁵I-Tyr¹¹-SS-14 demonstrated a significant reduction in binding density in the denervated parts of the neocortex. The decrease in specific binding was most pronounced (40–50%) in the superficial layers (I–III) of the frontal, parietal and temporal cortices 2 and 4 weeks after lesion. A significant loss in¹²⁵I-Tyr¹¹-SS-14 binding in the deeper layers was only observed in the frontal cortex after 2 and 4 weeks. In the occipital cortex a significant decrease was measured in the superficial layers only after 4 weeks. The specific binding in all cortical regions returned to normal after 6 weeks. The results suggested that¹²⁵I-Tyr¹¹-SS-14 binding sites are localized on cholinergic afferents in the rat neocortex and that an up-regulation of number of binding sites, alternatively an increased binding affinity occured with time after lesion.     

SPECT imaging of cerebral blood flow changes induced by acute trigeminal nerve stimulation in drug-resistant epilepsy. A pilot study

ObjectiveTo explore the cortical areas targeted by acute transcutaneous trigeminal nerve stimulation (TNS) in patients with drug-resistant epilepsy (DRE) using single photon emission computed tomography (SPECT).MethodsTen patients with DRE underwent brain SPECT at baseline and immediately after a 20-minute TNS (0.25 ms; 120 Hz; 30 s ON and 30 s OFF) applied bilaterally to the infraorbital nerve. The French Color Standard International Scale was used for qualitative analyses and z-scores were used to calculate the Odds Ratio (OR).ResultsAt baseline global hypoperfusion (mainly in temporo-mesial, temporo-parietal and fronto-temporal and temporo-occipital areas) was detected in all patients. Following TNS, a global increase in cortical tracer uptake and a significant decrease in median hypoperfusion score were observed. A significant effect favoring a general TNS-induced increase in cortical perfusion (OR = 4.96; p = 0.0005) was detected in 70% of cases, with significant effects in the limbic (p = 0.003) and temporal (p = 0.003) lobes. Quantitative analyses of z-scores confirmed significant TNS-induced increases in perfusion in the temporal (+0.59 SDs; p = 0.001), and limbic (+0.43 SDs; p = 0.03) lobes.ConclusionShort-term TNS is followed a global increase in cortical perfusion, namely in the temporal and limbic lobes.SignificanceThe TNS-induced perfusion increase may reflect neurons’ activity changes in cortical areas implicated in the epilepsy network.     

Layer-specific dilation of penetrating arteries induced by stimulation of the nucleus basalis of Meynert in the mouse frontal cortex

To clarify mechanisms through which activation of the nucleus basalis of Meynert (NBM) increases cerebral cortical blood flow, we examined whether cortical parenchymal arteries dilate during NBM stimulation in anesthetized mice. We used two-photon microscopy to measure the diameter of single penetrating arteries at different depths (∼800 μm, layers I to V) of the frontal cortex, and examined changes in the diameter during focal electrical stimulation of the NBM (0.5 ms at 30 to 50 μA and 50 Hz) and hypercapnia (3% CO₂ inhalation). Stimulation of the NBM caused diameter of penetrating arteries to increase by 9% to 13% of the prestimulus diameter throughout the different layers of the cortex, except at the cortical surface and upper part of layer V, where the diameter of penetrating arteries increased only slightly during NBM stimulation. Hypercapnia caused obvious dilation of the penetrating arteries in all cortical layers, including the surface arteries. The diameters began to increase within 1 second after the onset of NBM stimulation in the upper cortical layers, and later in lower layers. Our results indicate that activation of the NBM dilates cortical penetrating arteries in a layer-specific manner in magnitude and latency, presumably related to the density of cholinergic nerve terminals from the NBM.