Avian nocturnal hyperactivity: Neurotransmitter metabolism in the basal ganglia

B&n Rt~stwrth B~lletirl, Vol. 5, Suppl. 2, pp. X%374. Printed in the U.S.A

Avian Nocturnal Hyperactivity: Neurotransmitter Metabolism in the Basal

Ganglia’

DONALD KAY RIKER, BARRY I. GOLD2 AND ROBERT H. ROTH

Departments of Pharmacology and Psychiatry, Yale University School of Medicine, New Haven, CT 06510

RIKER, D. K.. B. I. GOLD AND R. H. ROTH. A&m notturnul hvperwtivity: N~~r~~~~uns~iff~r ~etub~~iis~ in the basal

gangfiu. BRAIN RES. BULL. 5: Suppl. 2,369-374, 1980.-The re$onaI dis~~bution of GABA, GAD activity, high-unity 3H-choline accumulation (HACA), and the dopamine/serotonin metabolites 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) are described in the brain of a migratory passerine species, the White-throated Sparrow (Zonotrichia albicollis). GABA and GAD activity are highest in the optic tectum and basal ganglia (Iobus paraolfactorius and paleostriatum). A high HACA rate and DOPAC-HVA level is restricted to the basal gang&a. The mesenceph~on has the highest 5-HIAA content. Avian nocturnal activity (ANH), a seasonal behavior of migratory passerines in captivity, is a manifestation of migratory flight on spring and fail nights. Dopamine metabolite levels and HACA during spring ANH were compared to two groups of birds rendered inactive at night. HVA and DOPAC in the basal ganglia were greater (+31 to 68%) in the hyperactive group. Yet, despite hyperactivity, the HACA rate remained unchanged in these regions. These data are consistent with a role for meso-telencephalic dopamine neurons in the release of ANH.

High-affinity 3H-choline accumulation Homovanillic acid Dihydroxyphenylacetic acid S-hydroxyindoleacetic acid Glutamic acid decarboxylase GABA White-throated Sparrow Locomotor activity Dopamine Acetylcholine Paleostriatum Paraolfactory lobe Basal gangiia Hyperactivity

DURING the last two decades investigators of neuro- t~nsmitter systems have probed brainstem and forebrain in an effort to explain the adaptiveness and mechanism of altered behavior. Most often the behavior of motor or endocrine outputs are described or manipulated. However, often these behaviors are born of convenience, or at least the adaptive significance of species-specific behavior is not considered.

One of us (DKR) has been investigating a naturally- occurring, nocturnal hyperactivity state manifest by migratory passerine birds [26-287. Avian nocturnal hyperactivity (ANH or Zugunruhe) occurs only on spring and fall nights, not in the winter or during periods of molt. Its annual organization is predictable and is thought to reflect a circannual rhythm entrained by photoperiod. The behavioral signs of ANH-hyperkinesia, stereotypic postures and movements, and bursting forelimb tremor-and the development of striatal, not iso-cortical, motor systems during the encepha- lization of the avian brain [14, 15, 21, 241 suggest that this spontaneous motor behavior is modulated by the basal ganglia. The adaptive significance of ANH, a behavior defined in captivity, is understood to be a reflection of normal migratory flight which occurs on spring and fall nights. Moreover, the

behavioral signs elicited by captivity may relate to normal components of migrato~ night flight-for example, hyperkinesia to non-stop flight, stereotyped posturing to pre-flight intention and bursting forelimb tremor to so-called “flap- glide” flight patterns specific to migration.

In this report we first present an analysis of regional distribution of neurotransmitter markers in the White-throated Sparrow brain. The White-throated Sparrow (WTS; Passer- iformes: Fringillidae: Zonotrichia albicoflis) is a North American temperate zone migrant. Zonotrichia species are often the subject of neuroendoe~ne and behavioral investigations 119,341. However, little attention has been directed to the neurochemistry of the passerine brain with the excep- tion of the hypothalamus [23]. Second, we investigated high&inity 3H-choline accumulation and steady-state levels of dopamine metabofites in the basal ganglia during ANH.

METHOD

Behmiord Ass~~ssnrent of Activity at Night

WTS’s caught in the fall were housed in a large aviary then transferred a few days before experiments to locomotor activity cages designed to monitor perch-related activity. A

*Supported by NINCDS National Research Service Award no. NS-05406 (DKR) and USPHS no. MH-14092 (RHR) and the State of Connecticut.

“Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20014.

Copyright @ 1980 ANKHO International Inc.-0361 -9230/80/080369-06$01.10/O

370 RlKER, GOLD AND ROTH

digital-to-analogue converter was designed to output con- tinuous on-line histograms of perch activity (15 minibar) to a Gould Brush recorder (Riker and Davis, in preparation). Cages were housed in a lightproof chamber with main fluorescent lights (2 x 40 W, - 150 lux in cage) and incandes- cent twilight lights (2 x 7.5 W, 3 lux in cage). Dim, diffuse night lights were provided (co.01 lux in cage). Photoperiods remained fixed during experiments and included 30 mm dawn and dusk periods (12D:OJT: ilL:OST). For detailed discussion of methods and circannual variation of ANH in the WTS see Riker 1261.

Producing NocrEtrnali~-inactive Birds in the Spring

In order to generate a group of inactive birds in the spring contemporaneous with normally hyperactive birds we em- ployed two lighting manipulations known to suppress ANH.

Prolongution of dusk illumination: Two light intensity thresholds must be passed during dusk before ANH can begin [26]. The first (-50 lux) encourages roosting; the second (- 1 lux) signals the start of night. If twilight (-3-5 lux) is artificially prolonged roosting continues through the night and normal ANH is never expressed.

Ab~~~l~te darkness: Very low-level night lighting (<O.Ol lux) is required for expression of ANH since absolute darkness in not encountered in the wild. Removal of all light constrains all movement at night.

Bruin Dissection

Birds were removed from activity cages and were quickly sacrificed in the dark by decapitation 4-5 hr after dusk. Brains were rapidly removed (-30 set), placed in sahne (O.%, i”), and transferred to a custom-made Plexiglas brain mold immersed in saline (1”). The brain rests ventral surface up. Thin grooves (0.25 mm) traverse the anterior-posterior axis of the mold (or brain) every millimeter (rostral pole=0 mm). Razor blades were inserted in the 2nd, Sth, 8th (A-+P) grooves. The two 3 mm slices (2-5, 5-8 mm) were transferred caudal side up to a chilled ice platform. Viewed through a dissecting microscope the basal and dorsal por- tions of each forebrain slice were blunt dissected along the lamina medullaris dorsalis. Lobus paraolfactorius (LPO) and olfactory tubercle comprise the basal portion of slice 2-5 mm. The paleostriatum primitivum, paleostriatum augmentatum (PA) and nucleus intrapeduncularis comprise the basal portion of slice 5-8 mm (PAL). The dorsal portion of slice 5-8 mm with the septum removed contains the neostriatum, ectostriatum and hyperstriatum ventrale and is termed “neo-hyperstriatum” (NH). The remaining caudal pole of the forebrain (8 mm-+posterior pole) is termed “caudal pole” and includes the archistriatum, posterior neostriatum and parahippocampus. The mesencephalic tegmentum is the entire mesencephalon without the optic tecta. Tissues for high-affinity :“H-choline accumulation were immediately assayed; tissues for other analyses were frozen at -70” until assay.

GABA Levels and Gltttamic Acid Decarboxylase Activity

GABA was measured in individual brain regions and whole brains of birds killed by decapitation. GABA was assayed by the radioreceptor assay of Enna and Snyder [ 121.

Glutamic acid decarboxylase (GAD) activitv was determined in the 57,000 g supernate incubated at 37jin imidazole acetic acid buffer (100 mM, pH 7.4, 20”) in the presence of 0.5 mM dithiothreitol, 0.5% v/v Triton X-100; 10.0 mM L- (f-‘C)glutamate and 50 PM pyridoxal phosphate. ‘YYO, was trapped on hyamine-soaked filter paper wicks. “Basal” GAD activity was defined as the specific activity at saturat- ing substrate concentration (10 mM glut~ate). The K, and V,,, of GAD was estimated by Eadie-Hofstee analysis (v vs. v/s) of reaction velocity at differring glutamate concentration (0.2-2 mM).

Whole brain ~-hydroxybutyrate (GHB) activity was measured by the GC method of Ando et a! 121.

High-ajjinity :‘H-choline Auwmulution in Bruin Synaptosomes

Individual brain regions, chilled in saline (1”) were homogenized in 0.32 M sucrose. High-affinity JH-choline accumulation (HACA) was measured in vitro in synaptosomes of the crude mitochondrial fraction (P,) as described by Riker et rrl [29]. “Basal” HACA was measured at 0.04 FM choline (4 min at 37°C) and compared to 1” blanks. The KT and V,,, of HACA was estimated by Lineweaver-Burk analysis at varying choline concentrations (0.1-0.55 PM).

D(l~urnin~l and Serofonin Me~aboiites fHVA, DOPAC and S-HIAA]

Dopamine metabolites (HVA, DOPAC) and the serotonin metabolite 5-HIAA were separated by gas chromatography and assayed by selected-ion monitoring mass spectrometry as described by Bacopoulos et ul [5,6]. Deuterated internal standards were added to correct for recovery. The data are presented in terms of unconjugated metabolites.

RESULTS

GABA and GAD Disfribution in Avian Brain

The WTS whole brain is GABA-rich (4.50 +moles/g wet wt, n=5 j-about 155% of the concentration reported for rat brain [8]. GHB levels in whole brain were about one thousand fold less than that of endogenous GABA (4.28 nmolesig, n=9) and twice that of rat (2.06 nmoleslg, 11 I]). This is the same GABA to GHB ratio as in rat whole brain.

The regional distribution of GABA and GAD in the WTS brain is shown in Table 1. The optic tectum has the highest level of both GABA (5.61 pmoleslg) and GAD (831 nmolesl mg protein/l5 min) followed by the lobus p~ao~acto~us- paleostriatum (5.03 pmoleslg; 569-488 nmolesimg protein’15 min), and diencephalon (4.46 pmoles/ g; 520 nmules/mg proteinll5 min). The medulla-pons, cerebellum, neo- hyperst~atum, and “caudal pole” were low in GAD activity and GABA. The GABA dist~bu~~on within the forebrain is not entirely homogeneous. The basal forebrain has two-fold higher levels of GABA than its dorsal counterpart. Basal slices (2mm) in the more rostral LPO are 32% higher than paleostriatal slices.

The avian midbrain tegmentum, an area which receives descending afferents from the LPO and PA 1171, has higher GABA levels than predicted by the rank-order of GAD activity. The Spearman rank correlation between GAD activity and GABA levels is i-,=0.&4 and increases to r,=0.83 when the midbrain tegmentum is not considered. These correla- tions suggests that, in general, GABA and GAD covary and

NEUROTRANSMITTERS AND AVIAN NOCTURNAL ACTIVITY 371

TABLE 1 REGIONAL DISTRIBUTION OF NEUROTRANSMI’ITER MARKERS IN THE BRAIN OF THE WHITE-THROATED SPARROW

(Zonorrkhio albicoilis)

GABA GAD HACA DOPAC HVA S-HIAA cLmoles/g nmoles CO,/ pmimg pro/4 ngig ngig rig/g

mg pro/l5 min min (n=5-11) (n=6) (n=3) (n= 1 l-26) (n= 1 l-26) (n=%25)

Te~enceph~on Lobus paraolfactorius*

\

P~eost~atum* Caudal pole Neo-hyperstriatum

Diencephalon Thalamus-hypothalamus

Mesencephalon Optic tectum Tegmentumt

Cerebellum

Medulla-ponsi

5.03 r?r 0.23

- 3.09 r 0.23

4.46 t 0.28

5.61 F 0.42 5.33 rt 0.23

2.64 t 0.25

3.26 + 0.25

569 -+ 31 8.42 2: 0.49

488 -+ 79 7.51 r!z 0.74 371 i 27 -_

430 + 35 1.81 Lf 0.13

520 -t 86 1.99 2: 0.29

831 r+ 54 1.00 -‘I 0.12 412 -+ 77 I.79 2 0.21

382 f 43 1.37 f 0.54

361 -t 31 0.96 :‘: 0.22

689 -t 56

378 5 34 51% 2 23-c 3

-

-

112t 5

-

-

478 + 48

313 t: 29 63rt 3 202 2

-

-

812 5

-

35s -+ 34

410 c 35 318 t 22 259 rt 21

-

-

709 t 57

-

-

Values are mean C SEM of birds sacrificed mid-night. The rank order of GAD, HACA, HVA, and 5-HIAA in the four telencephaiic regions and the mesencephalic tegmentum show some concordance (Kendall’s coefftcient of concordance, W=O.59, ~~0.05) due to the high values of HACA, GAD and RVA in the LPG and PAL.

*Widespread, green glyoxylic acid histofluorescence typical of catecholamine nerve terminals (Riker, Bunney and Roth, unpublished observations). tCreen histofluorescence cell groupings present (ibid). Per gram=gram tissue (wet weight). GABA=?-aminobutyric acid; GAD=glutamic acid decarboxylase; HACA=high affinity 3H-chotine accumulation; DOPAC=free dihydroxyphenylacetic acid; HVA=free homovanillic acid; S-HIAA=free 5-hydroxyindoleacetic acid. See METHOD for explanation of anatomical dissections.

that midbrain nuclei may receive extensive GABAergic innervation.

We determined the Km and V,,, of GAD in the two GABA-rich regions, PAL and optic tectum, and the neo- hyperstriatum. No differences were seen between these three regions (Km-3 mM; V,,,-250 nmoles/mg pro/l5 minf. In some cases kinetic plots revealed two separate lines which taken together deviate from Michaelis-Menton kinetics.

High-affinity :%i-cholitw Accumulation in WTS Brain

Sodium-dependent high-af~nity %-choline accumuIation (HACA) in rat hippocampal synaptosomes is thought to be the rate-limiting step in the synthesis of acetylchotine in nerve endings and is coupled to neuronal activity 1201. In the current study we attempted to use this variable as a marker for cholinergic nerve terminals.

HACA is strikingly specific to the avian basal forebrain. (LPO and PAL, Table 1). The entire brainstem and cerebellum have low accumulation rates (0.96-1.99 pmimg pro/4 min); further HACA in the NH is low (1.81 pmmg pro/4 min). In sharp contrast, HACA in the LPO and PAL is 4-8 fold higher than these regions. The distribution of HACA in 2 mm basal forebrain slices indicates that the paleostriatum (5-8 mm) has approximately twice the HACA rate of the more rostra1 LPO (2-5 mm) (Riker and Roth, unpublished observations).

The Kr and V,,, of HACA in the WTS forebrain was determined on several winter nights during nocturnal quies- cence. The basal forebrain areas (LPO and PAL) have a comparabte V,,, (104 pmlmg pro/4 min). The LPO has a 37%

higher K, (0.73 vs l.OO@M) than the PAL. The KT of the NH (1.04 PM) is comparable to the PAL, but the V,,,, is low (23 pmimg pro/4 min). The V,,, of the PAL and the NH are equivalent to those reported [29] from homologous areas of the cat brain (caudate-putamen, 124 pmmg pro/4 min; neocortex, 24 pm/mg pro/4 min).

Regional Distribution of Dopamine and Serotonin Metabolites (HVA, DOPAC and 5-HIAA)

Steady-state levels of DA and 5-HT metabolites in brain reveal not only the presence of these chemic~ly-de~ned neurons, but fluctuations in these levels provide a useful index of alterations in their physiological activity [ 18,301. We measured HVA, DOPAC and 5-HIAA in order to determine the distribution of dop~inergic and serotoneric neurons in WTS brain and, second, to investigate changes in transmitter metabolism during ANH.

HVA, DOPAC and 5-HIAA levels of four forebrain regions and the mesencephalon of the WTS are shown in Table 1. DA metabolites (DOPAC and HVA) are highest in the LPO and are about 40% lower in the paleostriatum. The dorsal (neo-hyperstriatum) and caudal regions (“caudal pole”) of the forebrain contain almost no DA metabolites. The mesencephalic tegmentum contains the DA cell bodies of the nucleus tegmenti pedunculo-pontinis, pars compacta (NTPC) which project to the paleostriatum augmentatum [13,17] and has intermediate metabolite levels (112 rig/g DOPAC; 81 ngig HVA).

All four forebrain regions have more uniform levels of the 5-HT metabohte 5-HIAA (259-410 r&g); the NH has the

RLKER, GOLD AND ROTH

FIG. 1. Steady-state levels of Free Dopamine Metabolites and High-atinity 3H-choline Accumulation Compared in the Basal Forebrain of Nocturnally-active and Nocturnally-inactive White- throated Sparrows (Z. albicollis) in the Spring. O=Noctumally- active White-throated Sparrows (Z. alhicollis) sacrificed 45 hours after dusk in the spring. In the DOPAC-HVA comparison the HVA values appear to the right; the active birds’ HVA values are further denoted by striped bars. k?l=Previously-active birds rendered inactive by 45 hrs of extended dusk twiIi~t and sacrificed. l =~evious~y-active birds rendered inactive by 4-S hrs of absolute darkness and sacrificed. Values are mean percent change relative to nocturnally-active (normal) spring birds + SEM (%). LPO=Lobus paraolfactorius and olfactory tubercle; PAL=paleostriatum primitivum and augmentatum. *p<O.OS; **p<O.O2, Mann-Whitney U-test, two-tailed; other comparisons, p>O.OS.

lowest concentration. The mesencephalic tegmentum, which in mammals receives raphe innervation [25], has twice the S-HIA_A_ content of the forebrain (709 r&g).

Dopamine ~eta~o~jte Levels ~urifl~ nocturnal hyperactivity

Steady-state levels of DA metabolites (DOPAC and HVA in the basal ganglia (LPO and PAL) were assessed in the spring 4-5 hr after dusk. Normally the rate of spring ANH reaches its peak during this time [26]; in addition, 4 or 5 hours should allow sufftcient time for DA metabolites to reach a new steady-state. NocturnaIly-inactive birds were generated by either prolonging twilight or maintaining absolute darkness as described in METHOD. All birds had demonstrated ANH on nights previous to the experiment.

DOPAC and HVA levels were consistently decreased in both the LPO and PAL of birds in either inactive group (Fig. 1). However, given these sample sizes decreases in HVA gained significance more often. The “absolute darkness” group had significantly lower HVA levels than the “actives” in the PAL and LPO (-41%, ~~0.02; -33%, ~~0.05 Mann-Whitney U-test, two-tailed). Interestingly, decreases in HVA in both these basal ganglia are twice those of the “extended twilight” group. Alterations in DOPAC levels appeared somewhat less reliable as an indicator of nocturnal activity. The DOPAC and HVA levels of the LPO, a ~at~~hola~ne-~ch area of undefined relationship to motor output, are less well correlated to ANH. In particular, no signiticant differences were found between HVA or DOPAC in the LPO of “active” and “extended twilight” groups. On the contrary, the PAL, the dopamine-rich area through which almost all forebrain motor output of birds must pass [9, 16, 171, has clearly lower HVA levels. Since other data indicate that the serotonergic metabolite, 5-HIAA, is significantly depressed in both the LPO and PAL (Riker and Roth, unpublished data) we do not want to suggest that the meso- limbic areas of the rostral forebrain (i.e.-LPO) are not active in the modulation of this behavior. However, DA me-

tabolism of meso-striatal neurons innervating the paleostriatum augmentatum appears to be reliably increased during ANH.

High-affinity “H-choline Accumulation During Nocturnul Hyperactivity

Since previous studies have demonstrated that in situ electrical stimulation of the septum [33] or in vitro depolari- zation of synaptosomes [22] increase HACA in the rat hippocampus we attempted to determine whether HACA varies with the dramatic behavioral changes observed in ANH. Thus, we compared HACA in basal ganglia (LPO and PAL) of WTS’s on spring nights to that of birds rendered inactive by either extending dusk twilight or maintaining absolute darkness as described in METHOD. Figure I shows that, contrary to the demonstrable changes in DA metabolites during ANH, HACA rate (at 0.04 PM choline) is not differ- ent in either the LPO or PAL of active or inactive groups. HACA does not appear to reflect the increased activity of dop~iner~c afferents to these cholinergi~~ly-innervated regions of the telencephalon, nor does HACA reflect the hyperactivity of these animals.

DISCUSSION

The neuroanatomy of the avian basal ganglia has many homologies with mammalian meso-telencephalic organization. Reciprocal connections between the NTPC and PA of the pigeon contan an ascending dopaminergic limb homologous to the nigro-striatal pathway [ 13, 171. In the avian forebrain, perhaps more so than the rn~rn~i~, many dorsal cortical nuclei must project to the PA synapse on neurons which project to the paleostriatum primitivum (pallidum) to gain exit to thalamic and brainstem nuclei.

Our findings in a passerine species, the WTS, accord with both neuroanatomic and neurochemical investigations of pigeon and chicken. The paleostriatum augmentatum is highly fluorescent and contains a dense cholinergic and dopaminergic innervation (i.e., high HACA, DOPAC and HVA). DA (-3-4 pg/g, [26]) and free DOPACYHVA (- 300-700 r&g) concentrations in the paleostriatal complex are lower than rat striata f5], but much hi&her than ~~aceous species (e.g. chicken, quail; Riker and Roth, unpu~~sh~ observations) or pigeon [l]. The LPO, also highly fluorescent, has yet higher levels of DA metabolites (-400-1000 rig/g)) suggesting a dense dopaminergic innervation. The ratio of free DOPAC to HVA in WTS brain is approximately 1: 1 and therefore similar to rat [5]. In contrast cat (Riker, D. K., Riker, W. F. and Roth, unpublished observations), monkey, and human 161 have a preponderance of HVA in brain and CSF (5-30 x DOPAC). In contrast to the basal ganglia, dorsal and caudal areas of the forebrain have sparse histofluorescence, low levels of DA metabolites, and low HACA activity. All forebrain areas contain a significant level of S-HIAA, suggesting widespread innervation from 5-HT brainstem nuclei as described for rat [4].

Thus, the LPO and PAL receive a dense dopaminergic innervation, presumably from the NTPC. The high V,,, of HACA, comparable to cat caudate-putamen 1281, suggest that cholinerglc neurons are present at high density in both the LPO and PAL. Since it is known that the mammalian striatum is made up largely of intrinsic cholinergic interneurons our evidence in the WTS suggests that these avian homologues may also contain cholinerglc interneurons.

NEUROTRANSMITTERS AND AVIAN NOCTURNAL ACTIVITY 373

Thus, our results agree with the present conception of both avian and mammalian basal ganglia.

GABA-GAD activity is highest in the optic tectum which agrees with many previous studies on the avian optic tectum [7,14]. Less is known about GABA’s role in the avian basal ganglia. Our data demonstrate GABA is high in the LPO, PAL and midbrain tegmentum. Further, GAD is very high in the LPO, high in the PAL, and unexpectedly low in the midbrain tegmentum. Riker, Gold and Roth (unpublished observations) have shown that GABA levels do not show day-night variation in any region of the WTS brain in the winter.

Previous data on the neuropharmacology of ANH argue for a role of catecholamines in the release of ANH. Haloperidol, fluphenazine, and cr-methyl-p-tyrosine all suppress ANH [26]. The antipsychotics reduce the intensity, but not the pattern, of ANH. These findings suggest that dopamine receptor blockade or a reduction in dopamine synthesis rate reduce or abolish ANH. The reduction we observed in steady-state levels of DA metabolites (DOPAC and HVA) in the PAL of previously-active spring birds rendered inactive at night supports the contention that DA metabolism is elevated during ANH and is reduced by inactivity. Taken together these data strongly suggest that increased activity of DA afferents to the PA releases ANH.

Increased DA metabolism in the basal ganglia is probably directly related to the expression of ANH. WTS’s rendered inactive by prolonging dusk twilight never receive the cue to commence nighttime activity (- 1 lux). They continue to roost and ANH is inhibited. On the other hand, WTS’s main- tained in absolute darkness have received the two cues at dusk necessary to commence ANH. Therefore, ANH in this group is consrruined by darkness, not inhibited. Since both these inactive groups demonstrate reductions in DA metabolite levels with respect to hyperactives it is not a simple day-night difference that separates actives from inactives. It

must be concluded that the release and expression of ANH, not the animal’s underlying physiological status, is correlated with increased DA metabolism in the basal ganglia.

Despite the clear alteration in behavior and DA metabolism in the basal ganglia during ANH we could detect no change in HACA in these same regions. Much recent work on HACA has documented that striatal HACA, in sharp contrast to hippocampal HACA, does not reflect change in dopaminergic afferent activity and responds differently to drugs [ 3, 29, 3 1, 321. In particular, dopamine receptor block- ers which increase ACh levels and turnover in the rat sttiatum do not alter HACA 1311. Increases in nocturnal locomotor activity and DA metabolism in the avian LPO and PAL without concurrent change in HACA strongly argue that paleostriatal HACA is not coupled to activity of these striatal motor pathways. Yet, the current negative findings do not allow conclusions to be drawn concerning change in the activity of cholinergic neurons within basal ganglia. This conclusion accords with the only behavioral investigation of HACA in which hippocampal, but not striatal. HACA was behaviorally altered in rats [lo].

We conclude that increased dopaminergic activity in meso-paleostriatal neurons of the WTS is directly responsi- ble for the expression of ANH and that this increased activity is not a reflection of nocturnal change unrelated to ANH. As previous studies had demonstrated pharmacologically, striatal HACA appears an insensitive measure of neuronal activity in basal ganglia pathways.

ACKNOWLEDGEMENTS

We thank Dr. R. Leslie Choi, Dr. Noriko Ando and Ms. Jan Abele for participating in the assay of brain tissue. Mr. Malcolm Lerch of Penn Yan, New York and Mr. Fred Sibley of Yale Univer- sity provided field assistance in the capture of sparrows. We also thank Mrs. Lynn Williams for her excellent assistance in typing this manuscript.

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Avian nocturnal hyperactivity: Neurotransmitter metabolism in the basal ganglia