Regional cerebral palmitate incorporation after unilateral auditory deprivation in immature and adult Fischer-344 rats

EXPERIMENTAL

NEUROLOGY

lot),

49 1-505

(1988)

Regional Cerebral Palmitate Incorporation after Unilateral Auditory Deprivation in Immature and Adult Fischer-344 Rats OSAMUTONE,JOSEPHC.MILLER, JANEM.BELL, AND STANLEY I. RAPOPORT’ Laboratory of Neurosciences, National Institute on Aging, National Institutes of Health, Bethesda, Maryland 20892 Received August 31, 1987 Regional cerebral incorporation of intravenously injected [U-‘4C]palmitate was measured from 1 day to 13 weeks after left cochlear destruction in 1 l-day- and 3 month-old, awake Fischer-344 rats. In 1 l-day-old animals, statistically significant left-right differences in incorporation were absent 1 day after cochlear destruction and were found only in parts of the cochlear nucleus and inferior colliculus after 1 week. After 6 to 13 weeks, consistent with functional neuroanatomy of central auditory regions, incorporation was reduced by 6 to 9% in the left cochlear nucleus and left lateral superior olivary nucleus, compared with corresponding right-side regions. The right medial superior olivary nucleus, medial nucleus of the trapezoid body, lateral lemniscus nucleus, inferior colliculus, medial geniculate body, and auditory cortex had 5 to 9% less incorporation than did corresponding left-side regions. Fewer significant differences after chronic auditory deprivation occurred in 3-month-old rats than in 1 l-day-old rats following cochlear destruction. Reduced incorporation corresponded to reported changes in cell morphology, which also were greater in immature than mature rats following auditory deprivation. The results suggest that the palmitate method can be used to identify long-term regional changes in the turnover of brain lipids after sensory deprivation. 0 1988 Academic PI-W., Inc.

INTRODUCTION Auditory input from the cochlea is distributed to central ascending response-specific neuronal populations ( 16) and is considered to be predomiAbbreviations: D-day, W-week, M-month, N-nucleus, DG-2-deoxy-D-glucose, Cb, brain radioactivity, rCMbc-regional cerebral metabolic rates for glucose. ’ The authors thank Dr. Timothy T. Soncrant for adapting his computer program for densitometric analysis of autoradiography, and Ms. Denise M. Larson for her technical support. We also thank Dr. Jean M. Gnaedinger for helpful advice and suggestions.

491

0014-4886/88 $3.00

492

TONE ET AL. I

< LEFT p

< RIDHT '

I COCHLEA I

VESTIBULOCOCHLEAR NERVE * COCHLEAR NUCLEUS 4

i I I I

1 SUPERIOR OLIVE NUCLEUS TRAPEZOID BODY NUCLEUS I I I I

4 LATERAL LEMISCUS NUCLEUS s INFERIOR COLLICULUS * MEDIAL CENICULATE * AUDITORY CORTEX

FIG. I. Scheme of ascending auditory pathway.

nantly ipsilateral to the superior olivary complex and trapezoid body, but predominantly contralateral commencing with the lateral lemniscus (Fig. 1) (5, 10,22,23,28,35). However, the extent to which this is true anatomically and functionally is not agreed upon. To assess functional activities of central auditory structures following auditory deprivation, regional cerebral metabolic rates for glucose, ICM&~, measured with [2-14C]deoxy-D-glucose (DG) (24, 26, 35), have been determined following unilateral auditory deprivation. Because the changes in rCMRglc frequently are small compared with the large coefficient of variation, left-right differences in DG incorporation whose coefficients of variation are smaller have been used to demonstrate statistically significant central effects (11). DG incorporation was reduced in the contralateral inferior colliculus of the 2 1-day-old mouse to 60% of the ipsilateral value immediately after unilateral cochlear destruction, approximated the ipsilateral value by 40 days after deprivation, and was 20% above the ipsilateral value by 240 days (28). The delayed recovery was ascribed to new innervation of the contralateral inferior colliculus from the superior olive and lateral lemniscus (27). In addition, unilateral cochlear destruction in gerbils reduced ipsilaterally compared with contralateral DG incorporation by 19 to 2 1% in the anteroventral and dorsal cochlear nuclei, did not affect left-right differences in the superior olivary complex, reduced contralaterally compared with ipsilateral incorporation by 18 to 29% in nuclei of the lateral lemniscus and by 54% in the

PALMITATE

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493

inferior colliculus, but did not affect relative incorporation by the medial geniculate body or auditory cortex (35), which are part of the central auditory system (10). These effects occurred in animals maintained in complete silence, demonstrating high intrinsic functional activity of auditory nuclei. The absence of differences in DG incorporation at the medial geniculate body and auditory cortex following unilateral cochlea destruction in gerbils; the high intrinsic functional activity of central auditory nuclei; and elevated DG incorporation into the inferior colliculus of mice after chronic unilateral damage, despite evidence of altered cell structure (33), suggest that the DG method may be of limited use for examining acute effects of auditory input into some auditory regions and chronic effects of auditory input into others. Changes in cell morphology have been demonstrated in the ipsilateral cochlear nucleus and the contralateral medial nucleus of the trapezoid body and the central nucleus of the inferior colliculus, following chronic unilateral auditory deprivation in rats and mice (1, 3, 33), and would be expected to be accompanied by evidence of altered regional metabolism. The morphologic changes are greater in lo- to 12-day-old than in 16- to 24-day-old animals (1, 3, 33), reflecting the fact that cochlear function matures between 11 and 20 days of age in these rodents (2,4,3 1). Our laboratory has developed a quantitative autoradiographic technique to measure the regional rate of incorporation of plasma palmitate into brain (13), as an indicator of local turnover and synthesis of brain lipids. The method may have advantages over the DG method for examining structural changes in the central nervous system that follow sensory deprivation or insults causing long-term recovery processes. For example, 3 to 7 days after 5 min of bilateral carotid occlusion in awake gerbils, incorporation of plasma [‘4C]palmitate into the CA3 and CA4 pyramidal cell layers and the dentate gyrus of the hippocampus was elevated, indicative of increased lipid synthesis during recovery from an ischemic insult (30). DG measurements show no chronic differences in these regions after the same 5 min of carotid occlusion (26). The palmitate method involves the intravenous injection of [14C]palmitate and the determination of brain radioactivity by quantitative autoradiography after 4 h. At 4 h, brain radioactivity has reached a steady-state value which does not differ from the 20- to 24-h value, and therefore is considered to represent radioactivity which has been incorporated into a “stable” metabolic compartment (2 1). Indeed, brain radioactivity 4 h after i.v. injection of [U-14C]palmitate is found mainly in membrane lipids, and to some extent in proteins (8, 13, 17). We thought it of interest to use the [‘4C]palmitate method to examine effects on lipid turnover within central auditory structures, following acute

494

TONE

ET AL.

and chronic unilateral auditory deprivation in 1 l-day-old and adult 3month-old rats. An abstract of this work has been published (29). MATERIALS

AND

METHODS

Male Fischer-344 rats were obtained from Charles River Breeding Laboratories (Wilmington, MA) and were maintained on standard rat chow and water ad libitum. Eight- to ten-day-old pups were purchased with their mothers. Three series of experiments were done: (i) In 1 l-day-old rats ( 1 lD), the left cochlea was destroyed 11 days after birth, and incorporation of plasma palmitate into various brain regions was measured after 1 day ( 11D + 1D), lweek(llD+ lW),6weeks(llD+6W),or13weeks(llD+ 13W).(ii)In 3-month-old rats (3M), the left cochlea was destroyed 3 months after birth, and palmitate incorporation into brain was measured 1 day, 1,6, or 13 weeks later. (iii) Control rats were anesthetized with halothane 11 days after birth and palmitate incorporation was measured 6 weeks later. The left cochlea was destroyed with fine forceps, using a transmeatal approach in rats anesthetized with 1 to 2% halothane. Destruction was confirmed postmortem by direct observation with a dissecting microscope (9, 12); the right tympanic membrane remained intact. In separate experiments, the lack of a Preyer’s reflex (32) in rats bilaterally deprived by the same procedure crudely demonstrated that the animals were deaf. Sound levels of 50 to 70 dB were measured in the experimental environment (Model 886 Sound Level Meter, Simpson Electric Co. Elgin, IL). The method of Rimes et al. (13) was abbreviated to measure left-right differences in the regional rate of incorporation into brain of intravenously injected plasma [‘4C]palmitate. Polyethylene catheters (PE 50), filled with 100 IU Na heparin per milliliter 0.9% (w/v) NaCl, were inserted into the femoral artery and vein of a rat under 1 to 2% halothane anesthesia. The skin incision was sutured and infiltrated with xylocaine. The hindquarters of the rat were wrapped in a fast-setting plaster cast which was taped to a wood block, and the rat recovered from anesthesia for 4 h. The following parameters were measured immediately prior to [ 14C]palmitate injection: hematocrit value, heart rate, arterial blood pressure, blood pH, and blood gases. A solution of [‘4C]palmitate (uniformly labeled, 800 to 900 mCi/mmol, New England Nuclear, Boston, MA) in 5 mMHepes buffer, pH 7.4, containing 5 mg/ml bovine serum albumin, was injected into the femoral vein (450 j&i/kg). The rat was killed 4 h later by an overdose of Na pentobarbital. After decapitation, the brain was removed, frozen in 2-methylbutane cooled to -65°C and later sectioned on a cryostat at -20°C into 20-pm-thick coro-

PALMITATE

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DEPRIVATION

nal sections which were collected on glass coverslips and dried on a hot plate. The tissue sections and radioactive standards (8-208 nCi/g) were exposed 5 weeks to X-ray film (SB5, Kodak, Rochester, NY). After the tilm was developed, the autoradiographs were scanned with a microdensitometer (Model 700- 10-90, Gamma Scientific, San Diego, CA) (25). Optical density was converted to brain radioactivity, Cbr nCi/g, by means of a standard curve. Left-right differences (percentage) in Cb, were determined in 32 regions, including 13 from the auditory pathway (IO), and were calculated as follows, Percentage difference =

100

(Cbr,let?

-

cbr,right

)

Cbr,right

*

111

Greater left-side (ipsilateral) decreases in Cbr would yield negative values and greater right-side decreases would produce positive values. A normalized Cb, was calculated by dividing Cbr of a specific region by the average Cb, of 19 nonauditory regions in the same animal, giving the ratio Normalized

Cbr =

100 (Regional Cb,) Average nonauditory Cb, *

Comparison of experimental with control normalized Cbr)s indicated whether regional [i4C]palmitate incorporation was increased or decreased. The extent of cochlear destruction was determined 13 weeks after an operation in 1 l-day and in 3-month rats (n = 4). Under anesthesia, these rats were killed by transaortic perfusion with isotonic saline containing 10 III/ml heparin, followed by 200 ml 3% pamformaldehyde and 3% glutaraldehyde in 100 mJ4 cacodylate buffer, pH 7.4. The skull and brain were removed and stored overnight in the same fixative at 4°C. The skull was decalcified for 1 to 2 weeks in a 12% EDTA solution, pH 7.4, containing 5% sucrose. The tissues then were embedded in plastic, cut into 3-km sections, and stained with hematoxylin-eosin and toluidine blue. The cochlea and posterior ventral cochlear nuclei were examined by light microscopy and compared with sections from untreated 3-month-old rats (15, 19,34). The statistical significance of differences of mean values from zero was estimated by Bonferroni t statistics for multiple comparisons (18), whereas the significance of differences between experimental and control means was determined by Dunnett’s test for multiple comparisons (6). RESULTS Rats exhibited a Preyer’s reflex (32) from 12 days after birth; their eyes opened at 12 to 13 days. Body weight increased progressively with age (Table

496

TONE ET AL. TABLE 1 Physiological Parameters” llO+lO

llO+lW

3 wei!M

:i

::

Mean blood prorruro lm

::

66 *

:::

:i7

ND

ND

7.37 f0.01

fO.O1

Pai02 Hgl lml

ND

ND

Pa02 Inun Hg)

ND

ND

NO

ND

Pwamotors

Hoart Ibpml

mto

Arterial

37

tocrit

pti

tlgl

llD+6W

f;:;“,zl

E’ * ::”

21

*

llD113W

::i *

3M+lD

3M+lW

237 f9

2’

113

3W+6W

ii.’

120

3M113W

*

:“9”

:F

*a

fl lZO

:;*

9.2

::’

2f

*

465

211

426 27

:::

396 215

381

f10

::':

7.35

7.38

fO.O1

7.40 f0.03

7.39 f0.03

7.37

7.39

-If.: .

21.4 31.1

::*z .

il.7 29.3

_ . r,3*,7

::*: .

::*: .

::::

2:

X

::::

92.5 f3.D

::*: .

90.3 f1.8

22

46 fl

2:

*

ND ::

:i

::

f2

to.01

*

116 il

to.01

’ Values are means + SE (n = 3-9); statistics were by Bonferroni multiple comparison test (18). Abbreviations: 1 ID + 1D (1 W, 6W, or 13W)-left cochlea was destroyed 11 days after birth and [‘4C]palmitate incorporation was measured 1 day (1, 6, or 13 weeks) later; 3M + 1D (lW, 6W, or 13W)-left cochlea was destroyed 3 months after birth and [‘4C]palmitate incorporation was measured 1 day ( 1,6, or 13 weeks) later; 11D + 6W Control-sham operation 11 days after birth and [‘4C]palmitate incorporation was measured 6 weeks later. ND-not determined. * Differs significantly from mean at preceding age, P< 0.05. 7 Differs from “1lD + 6W, Control”, P < 0.05.

1). Mean arterial blood pressure increased from 12 days and reached an adult value by 6 weeks. Mean arterial blood pressure of the 1 l-day-old group, deprived for 6 weeks (11D + 6W), was lower (P < 0.05) than that of corresponding controls. Heart rate of the 11 D + 1W group was significantly higher than in other groups. No significant differences were measured in arterial blood gases, hematocrit values, or body temperatures, although these measurements were not feasible in the youngest animals. Mean values of the physiologic parameters agree with values reported elsewhere (14,20,28). Left-right differences in brain radioactivity (Eq. [ 11) corresponding to differences in [14C]palmitate incorporation were determined 1 day to 13 weeks after destruction of the left cochlea in 11 -day-old rats (Table 2). Statistically significant differences (Fig. 2) were absent 1 day after destruction ( 11D + 1D) and were found 1 week after deprivation (11 D + 1W) in the cochlear nuclei (-5.6 to -5.4%), but not in other regions examined. Six weeks after deprivation (11D + 6W), significant differences equaled -8.7 to -6.2% in

PALMITATE

AND AUDITORY

DEPRIVATION

497

TABLE 2 Percent Differences in Brain Radioactivity (Eq. [ 11)between Left and Right Regions after Left Cochlea Destruction in 11-Day-Old Rats”

-

(1.7

*

1.4

Ml

-

8.3

*

0.9

I”

-

6.2

f

1.1

l *

-

6.0

*

1.1

. .

6.2

t

1.P

8.6

t

2.0

l

8.6

*

1.4

Y

I.5

*

1.1

9.6

t

1.9

l *

7.7

f

1.9

l * l *

4.8

f

1.2

3.b

*

1.1

4.6

*

2.0

’ Values are means + SE (n = 8). Abbreviations: see Table 1, N-nucleus, Dors-dorsal, Vent-ventral, Post-posterior, Ant-anterior, Lat-lateral, Med-medial, Inf-inferior, C D-central dorsomedial, C V-central ventrolateral, Super-superticial layer, Deep-deep layer. * P < 0.05, **P < 0.01, differs significantly from zero (Bonferroni t statistics).

the cochlear nuclei, -6.0% in the lateral superior olivary nucleus, 8.4% in the medial trapezoid body nucleus, 8.6% in the lateral lemniscus nucleus, 9.4% in the central inferior colliculus, 7.7% in the medial geniculate body, and 4.8% in auditory cortex I. Similar but fewer significant left-right differences were found 13 weeks after deprivation ( 1 ID + 13W). None was found in sham-operated 11 -day-old controls deprived for 6 weeks ( 11D + 6W, control) or in nonauditory regions more frequently than expected by chance. Left cochlear destruction in adult, 3-month-old rats also produced leftright differences in [‘4C]palmitate incorporation into brain regions of the auditory pathway, but less frequently than in deprived 1 l-day-old animals (Table 3). No consistent changes were apparent in nonauditory regions. Comparison of normalized regional C,, values (Eq. [2]) in the deprived animals with the corresponding regional value in controls (Table 4) showed

498

TONE ET AL.

VA

FIG. 2. Autoradiographs of coronal brain sections 6 weeks after left cochlear destruction in I l-day-old rat. CNVA-anterior ventral cochlear nucleus, SONL-lateral nucleus of superior olive, LLN-nucleus of lateral lemniscus, GM-medial geniculate nucleus.

PALMITATE

AND AUDITORY

499

DEPRIVATION

TABLE 3 Percentage Differences in Brain Radioactivity (Eq. [ 11) between Left and Right Regions after Left Cochlea Destruction in J-Month-Old Rats” 3n

Auditory

+ 1~

3ll

3ti

+ IN

+ 6N

3M

+ 13W

Regions

Cochlear

N DJrr

- 4.4

* 0.8

- 4.9

2 1.5

Cochlsar

N vent

Port

-

3.9

2

1.3

-

7.3

f

2.6

Cofhloar

N vent

Ant

-

4.5

*

1.4

-

3.1

*

2.2

Swerior

Olive

N

L*t

-

-

8werior

Olive

N

Mod

Lst

Inf

Colliculus

Inf

Colliculur

Gmiculrto

- 1.4

1.6

2 0.8

*

1.3

-

5.6

2 1.1

-

1.6

t

1.5

1.1

t

-

*

1.3

4.0

*

0.5

1.1

* 1.4

1.6

2 1.3

*

1.3

3.0

*

1.7

1.5

f

1.4

1.3

f

1.3

4.8

*

1.7

2.5

2 1.9

5.1

2 1.8

4.1

*

1.0

l

3.8

f 1.1

8.0

f

1.7

6.5

* 1.4

7.3

f

2.1

l

C D

1.7

* 1.0

3.6

f

1.8

0.5

f

1.1

1.3

C V

3.8

2 1.1

7.4

2 1.2

l *

3.5

f

0.6

3.2

2 1.0 2 1.6

I.4

2 1.3

7.2

f

2.1

X

2.7

2 1.1

- 0.6

* 1.1

4.1

* 2.2

0.2

* 1.4

1.4

* 1.4

Mod

l

I

I*

-

36s

**

Auditory

Cortex

I

1.5

t

1.1

2.6

f

2.6

3.9

2 1.8

Auditory

Cortex

IV

3.5

2 1.1

3.3

2 2.5

3.3

2 0.9

Auditory

Cortex

V

0.7

*

0.6

0.7

* 2.0

4.1

2 2.4

0.3

t

0.9

- 1.1

f 1.3

-

2.4

2 1.3

0.4

2

1.3

f

1.2

-

4.9

f

-

0.8

2 1.2

-

1.4

2 0.8

-

Nm-Auditory

8

N

tfed

Vsstibular

N

Let

-

N

Lit

-

Csrebdlrr Lennircus

Wed

84m Colliculus

Svper

SW Colliculus Geniculote Lat

Deep Dar+

Cortex

-

-

I

csudate-Putamen somatose"rm-y

*

Regions

Vestibular

Visual

**

1.5

3.9

N

**

i 1.7

0.6

cl.4

7mpezoid8odyNlkl Lmi+cus

*I

cl-x

I

1.2

*

1.0

1.0

* 0.6

1.7

2 1.4

1.4

2 0.8

1.3

* 0.9

0.3

* 0.7

4.8

2 2.1

1.8

i

2.3

0.2

* 0.9

1.2

? 0.8

0.6

? 1.7

0.3

2 1.2

0.6

* 1.2

0.0

* 1.0

2.0

? 0.8

1.8

2 1.2

-

0.5

* 1.2

1.2

2 1.0

-

1.8

2 1.8

2.6

2 1.6

-

2.9

f

0.5

2 0.9

-

1.2

f

2.4

-

1.4

* 1.3

-

1.7

* 1.1

0.9

t

0.8

0.8

f

1.2

-

0.1

t

1.1

-

1.6

t

1.2

0.8

t

1.1

2.2

t

1.8

-

1.0

*

1.5

0.8

f

1.6

1.3

n Values are means + SE (n = 8). *P < 0.05, **P < 0.0 1, differs significantly from zero (Bonferroni f statistics). See Tables 1 and 2 for abbreviations.

that unilateral decreases in [‘4C]palmitate incorporation were produced in the ipsilateral left cochlear nuclei and lateral superior olivary nucleus, and in the contralateral right medial superior olivary nucleus, medial trapezoid body nucleus, and more cephalad auditory regions. These regions had lower ratios than either the control average or homologous regions in the same animal on the opposite side. For example, -8.7% to -6.2% lefkight differences in [14C]palmitate incorporation were found in the cochlear nuclei of the 11 D + 6W group (Table 2). These differences represented decreased leftside incorporation of isotope rather than increased right-side utilization (Table 4), because the normalized C, values for the right cochlear nuclei were not statistically different from control values. Incorporation decreased in

500

TONE ET AL. TABLE 4 Normahzed Regional Incorporation of [ “C]PaImitate into Brain (Eq. [2 J),6 Weeks after Left Cochlea Destrwtion in 11-Day-Old Rats” 110 + 6W Control

110 + 6W Left deprived Left

Average

Auditory Regions Coddear N Dors Cochlear

N Vent

Post

Cochlear N Vent Ant Superior Olive N Lat Superior Olive N Mad Trapezoid Body N t&d Lemniocus Lat N Inf Colliculur C 0 Inf Colliculus CV Geniculate Mad Auditory Cortex I Auditory Cortex IV Auditory Cortex V B Non-Auditory Regions Sup Colliculus Super Sup Colliculus Deep Geniculate Lat Oars Visual Cortex I

128 + 2

119 f 2 *

129

122

132 t 3

123 f 2 *I

131 i 2 f 1 131 f 2

131

2 2

124

f

2 *

131

125

f

121

f

2

1 ** 110 t 2 116 f 1 **

? 2

2

114 f 2

f

2 *

100 + 2 134 t 3 112 f 1 90 -+ 1 99 ?: 1 95 f 2

120 ?z 1 127 f 1 100 + 1 132 t 2 115 + 2 93 f 1 99 f 1 95 t 2

= 100) 102 f 1 98 k 1 107 f 1 102 t 1

101 98 106 102

124

(Average

Right

+ 2

f. t 2 f

1 2 2 1

133

114

99

:

2

+

f

2

120 i 106 f: 88t1 96 f 91 t

1 ** 1 **

100 96 104 100

1 1 2 1

i f i t

1 1

0 Values are means f SE (n = 8) of regional brain radioactivity divided by average brain radioactivity of 19 nonauditory regions (4 of 19 shown). *P < 0.05, **P < 0.0 1, differs significantly from control mean by Dunnett’s test (6).

contralateral right regions beyond the superior olive complex, from the medial superior olive to the medial geniculate (Fig. 1). Hematoxylin-eosin-stained sections from 11 -day-old rats deprived for 13 weeks ( 11D + 13W) showed gross destruction of the cochleae and degeneration of spiral ganglia (Fig. 3A-C). Toluidine blue-stained sections of posterior ventral cochlear nuclei showed no apparent degeneration (Fig. 3D-F) after 13 weeks, in either the 1 l-day-old or 3-month-old groups. Spherical cells in the 11 D + 13W group appeared smaller than in controls in agreement

PALMITATE

AND AUDITORY

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501

FIG. 3. Light micrographs of spiral ganglion and posterior ventral cochlear nucleus. A-Chematoxylin-eosin staining of spiral ganglion. D-F-toluidine blue staining of cochlear nucleus. A, D-control (3-month-old rat). B, E- 13 weeks after cochlear destruction in 1 l-dayold rat. C, F- 13 weeks after cochlear destruction of 3-month-old rat. Magnification, X50.

with a previous report ( 1, 3). No notable difference in cell size was observed after 13 weeks of deprivation in 3-month-old rats. DISCUSSION Chronic unilateral auditory deprivation, produced by destruction of the left cochlea, decreases incorporation of plasma [U-‘4C]palmitate into central

502

TONE

ET AL.

auditory regions of awake rats. Left-right differences in incorporation are consistent with functional neuroanatomy of ascending central auditory pathways and with reported effects of chronic unilateral deprivation on cell size in auditory nuclei (5, 10,22,27,33,35). Intravenously injected [U-i4C]palmitate which enters the brain undergoes several biochemical fates (8, 17). Tracer is esterified into complex lipids, primarily phosphatidylcholine and phosphatidylethanolamine; some is incorporated into protein. A third fate is @-oxidation followed by synthesis of aqueous metabolites, mainly [i4C]glutamate and [14C]aspartate, or lost as i4C02. Incorporation into lipids and proteins is rapid, and virtually complete within 15 min after i.v. injection. After 15 min, aqueous radioactivity continues to decline until at 4 h it equals 27% of net brain radioactivity. At 4 h, the lipid fraction comprises 60% of net radioactivity and the protein fraction comprises 15%. There is no significant difference between net brain radioactivity at 4 and 24 h, when aqueous radioactivity constitutes about 15% of the net. The rate of incorporation of plasma palmitate into the “stable” 4- or 24h compartment reflects turnover and synthesis of palmitate-containing brain structures (2 1). Reduced incorporation of [‘4C]palmitate into the “stable” 4-h brain compartment was found after left cochlea destruction in the ipsilateral, left cochlear nuclei (dorsal, ventral posterior, ventral anterior) and in the lateral superior olivary nucleus, indicating that auditory input to these nuclei is predominantly ipsilateral. On the other hand, reduced incorporation in the contralateral medial trapezoid body, and in the lateral lemniscus and the more cephalad auditory regions, suggests that these latter regions have predominant input from the contralateral ear. Left-right differences in incorporation following unilateral auditory deprivation correspond to reported effects on neuronal morphology in the ipsilateral cochlear nucleus, and the contralateral medial nucleus of the trapezoid body and central nucleus of the inferior colliculus, which are more evident in deprived immature animals than in mature animals (1, 3, 33). This correspondence provides further evidence that incorporation of plasma palmitate by brain reflects brain cell integrity and turnover of palmitate-containing lipids [see Introduction (2 l)]. Percentage differences in [ “C]palmitate incorporation between homologous left and right auditory regions, ranging from -8.7% to 9.4% (Table 2) following chronic left auditory deprivation in immature animals, are statistically significant throughout most of the ascending auditory system. These differences represent minimum unilateral decrements in incorporation, as auditory inputs are distributed bilaterally in all regions but the cochlear nuclei (7, 10, 16). Furthermore, reductions in incorporation into a specific lipid fraction or specific cell membrane may exceed the measured regional reduc-

PALMITATE

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tions, which average out unaffected as well as affected cellular components. Regional biochemical fractionation is necessary to address this issue. A significant decrease of [r4C]palmitate incorporation into most central auditory regions, with the exception of parts of the cochlear nucleus and contralateral inferior colliculus, is not found within 1 day or 1 week afier unilateral auditory deprivation in 11-day-old or 3-month-old rats, suggesting that plastic changes following acute reduction of neuronal activity require weeks to months to become fully evident. On the other hand, [14C]palmitate incorporation by the inferior colliculus remains low in chronically deprived rats, whereas DG incorporation in mice is elevated (27,28). These differences between the two tracers indicate that neuronal oxidative metabolism recovers and even becomes excessive following chronic deprivation, despite reduced turnover of membrane lipids and proteins, reduced cell size, and altered function in the affected regions (22,33). REFERENCES 1. BLATCHLEY, B. J., J. E. WILLIAMS, AND J. R. COLEMAN. 1983. Agedependent effects of acoustic deprivation on spherical cells of the rat anteroventral cochlear nucleus. Exp. Neurol. 80: 8 l-93. 2. BOSHER, S. K., AND R. L. WARREN. 1971. A study of the electrochemistry and osmotic relationships of the cocblear fluids in the neonatal rat at the time of development of the endocochlear potential. J. Physiol. (London) 212: 739-76 1. 3. COLEMAN, J. R., AND P. O’CONNOR. 1979. Effects of monaural and binaural sound deprivation on cell development in the anteroventral cochlear nucleus of rats. Exp. Neurol. 64: 553-566. 4. CROWLEY, D. E., AND M-C. HEPP-REYMOND. 1966. Development of cochlear function in the rat of the infant rat. J. Comp. Physiol. Psychol. 62: 427-432. 5. DIAMOND, I. T., M. FELDMAN, R. GALAMBOS, J. M. GOLDBERG, M. H. GOLDSTEIN, J. M. HARRISON, M. IGARASHI, B. W. KONIGSMARK, K. GSEN, G. RASMUSSEN, C. A. SMITH, H. H. SPOENDLIN, AND J. VAN NOORT. 1973. Neuroanatomy of the auditory system. Arch. Otolaryngol. 98: 397-4 13. 6. DUNNETT, C. W. 1964. New tables for multiple comparisons with a control. Biometrics 20: 482-49 1. 7. GLENDENNING, K. K., J. K. BRUNSO-BECHTHOLD, G. C. THOMPSON, AND R. B. MASTERSON. 198 1. Ascending auditory alferents to the nuclei of the lateral lemniscus. J. Comp. Neural. 197: 673-703. 8. GNAEDINGER, J. M., J. C. MILLER, C. H. LATKER, AND S. I. RAPOPORT. 1988. Cerebral metabolism of plasma “C-palmitate in awake, adult rat: Subcellular localization. Neurothem. Res., in press. 9. GREENE, E. C. 1963. Anatomy of the Rat. Hafner, New York. 10. HARRISON, J. M., AND M. E. HOWE. 1974. Anatomy of the afferent auditory nervous system of mammals. Pages 283-336 in W. D. KEIDEL and W. D. NEFF, Eds., Handbook of Sensory Physiology Vol. 5, No. 1. Springer-Verlag, Berlin. 11. HAXBY, J. V., R. DUARA, C. L. GRADY, N. R. CUTLER, AND S. I. RAPOPORT. 1985. Re-

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